Peptides comprising non-natural amino acids and methods of making and using the same

ABSTRACT

This invention relates to novel compositions comprising analogs of naturally occurring polypeptides, wherein the analog comprises an α-amino acid and at least one β-amino acid. Administration of the compositions may be used for effecting treatment or prevention of a plurality of disease states caused by dysfunctional biochemical or biological pathways. The compositions and methods of this invention are particularly useful to identify novel therapeutic modulators of in-vivo receptor activity with extended half-lives and relevant bioactivity as compared to the naturally translated polypeptides upon which the analogs are derived.

FIELD OF THE INVENTION

The invention relates generally to compositions comprising modifiedpolypeptide sequences with greater resistance to degradation andincreased receptor selectivity as compared to naturally encoded,unmodified polypeptide sequences, and to methods of making thecompositions and methods of using the compositions as pharmaceuticallyactive agents to treat disease in animals, including humans.

BACKGROUND OF THE INVENTION

The q25 region of chromosome 6 on the human genome encodes a VIP familymember that is 170 amino acids long which becomes post-translationallycleaved to form vasoactive intestinal peptide (VIP). The active form ofthe VIP polypeptide is a 28 amino acid protein that functions, amongother ways, to reduce arterial blood pressure, to increase vasodilationof blood vessel walls, to relax smooth muscle in the respiratory systemand gastrointestinal tissues, reduce inflammatory responses through bothpromotion of Th2 differentiation as well as the reduction of Th1responses, modulate both the innate and adaptive immune response, and tostimulate secretion of electrolytes in the gut. VIP has also been shownto be active in the central nervous system as a neurotransmitter and incommunication with lymphocytes.

VIP family members have short half-lives. For instance, VIP has ahalf-life of about two minutes in the blood stream. It is desirable toidentify polypeptides that mimic the function of VIPs such as VIP, buthave increased half-life and equivalent or more receptor selectivitythan the naturally occurring VIP amino acid sequence.

Bioactivity of VIP is transmuted through three known receptor subtypes:VIP₁R, VIP₂R, and PAC₁R. These receptors are known to induce cAMPconcentration as well as stimulate the production of intracellularcalcium. Their affinities for VIPs such as VIP vary depending upon thesubtype and the amino acid sequence of the ligand. VPAC1 has beenimplemented in cancer as well as inflammatory diseases such as multiplesclerosis, arthritis, parkinson's disease and alzhiemers. VPAC2Rdysfunction has been implemented in neurodegenerative disorders,diabetes, and pulmonary arterial hyerptsion (PAH), among otherdisorders. It desirable to identify a peptidomimetic of VIP to haveselectively antagonize or affect one VIP receptor subtype over anotherVIP receptor subtype in order to treat a disease related to thebiological affects of one receptor without disrupting or otherwiseinterfering with the normal biological affects of another receptor withthe same ligand.

SUMMARY OF THE INVENTION

The selected pattern of synthetic amino acids along the helicalpolypeptide decreases the rate at which the polypeptide may degrade whenadministered to a subject or when reconstituted or placed in solution.Selected side chains of the amino acids increase the conformationalrigidity of the polypeptide in order to constrain the polypeptide in itsactive state. The selected pattern of synthetic amino acids along thehelical polypeptide increases the half-life of the polypeptide ascompared to naturally encoded polypeptides with the same α-amino acidsequence. In some embodiments, the polypeptide comprises β-amino acidsthat spatially aligned along a longitudinal axis of the analog in orderto confer degradation resistance to the composition while preserving thenative binding interface. In some embodiments, the composition comprisesa VIP analog. In some embodiments, the composition comprises avasoactive intestinal peptide (VIP) analog based upon the sequencesdisclosed herein.

In some embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 10 percent toabout 60 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 12 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 14 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 16 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 18 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 20 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 30 percent toabout 50 percent of the total number of amino acids of the analog.

In some embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 40 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 45 percent toabout 50 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 40 percent toabout 45 percent of the total number of amino acids of the analog.

In some embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 30 percent toabout 40 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 35 percent toabout 40 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 20 percent toabout 30 percent of the total number of amino acids of the analog.

In some embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 10 percent toabout 20 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 15 percent toabout 20 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 20 percent toabout 25 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 25 percent toabout 30 percent of the total number of amino acids of the analog. Insome embodiments, the composition comprises a VIP analog wherein thetotal number of β-amino acids in the analog is from about 30 percent toabout 35 percent of the total number of amino acids of the analog.

Those of skill in the art will appreciate that amino acid positionscorresponding to positions in analogs can be readily identified in anyother molecule such as analog fusions, variants, fragments, etc. Forexample, sequence alignment by visual means or computer programs such asBLAST can be used to align and identify a particular position in aprotein that corresponds with a position in the analog of polypeptidesequences identified in this application or other GLP-1, VIP, PYY,IL-10, PACAP, Ghrelin, ANP/BNP/CNP, Maxadilan/M65,

The term analog encompasses polypeptides comprising one or more aminoacid substitutions, additions or deletions. Analogs of the presentinvention may be comprised of modifications with one or more naturalamino acids in conjunction with one or more non-natural amino acidmodification. Exemplary substitutions in a wide variety of amino acidpositions in naturally-occurring analogs have been described, includingbut not limited to substitutions that modulate one or more of thebiological activities of the analogs, such as but not limited to,increase agonist activity, increase solubility of the polypeptide,convert the polypeptide into an antagonist, decrease peptidase orprotease susceptibility, etc. and are encompassed by the term analog.

In some embodiments, the water soluble polymer is coupled to the GLP-1polypeptide at one or more of the amino acid positions: 19, 23, 26, 27,30, and 33 of the consensus sequence identified in Table 4.

In some embodiments, the analogs further comprise an addition,substitution or deletion that modulates biological activity of theanalogs. For example, the additions, substitution or deletions maymodulate one or more properties or activities of the analog. Forexample, the additions, substitutions or deletions may modulate affinityfor the analog receptor or binding partner, modulate (including but notlimited to, increases or decreases) receptor dimerization, stabilizereceptor dimers, modulate the conformation or one or more biologicalactivities of a binding partner, modulate circulating half-life,modulate therapeutic half-life, modulate stability of the polypeptide,modulate cleavage by peptidases or proteases, modulate dose, modulaterelease or bio-availability, facilitate purification, or improve oralter a particular route of administration. Similarly, analogs of thepresent invention may comprise protease cleavage sequences, reactivegroups, antibody-binding domains (including but not limited to, FLAG orpoly-His) or other affinity based sequences (including but not limitedto, FLAG, poly-His, GST, etc.) or linked molecules (including but notlimited to, biotin) that improve detection (including but not limitedto, GFP), purification or other traits of the polypeptide.

Vasoactive intestinal peptides (VIP/PHI) including, but not limited to,VIP, human, porcine, rat, ovine; VIP-Gly-Lys-Arg-NH₂; biotinyl-PHI(biotinyl-PHI-27), porcine; {Glp¹⁶} VIP 16-28, porcine; PHI (PHI-27),porcine; PHI (PHI-27), rat; PHM-27 (PHI), human; prepro VIP 81-122,human; prepro VIP/PHM 111-122; prepro VIP/PHM 156-170; biotinyl-PHM-27(biotinyl-PHI), human; vasoactive intestinal contractor(endothelin-beta); vasoactive intestinal octacosa-peptide, chicken;vasoactive intestinal peptide, guinea pig; biotinyl-VIP, human, porcine,rat; vasoactive intestinal peptide 1-12, human, porcine, rat; vasoactiveintestinal peptide 10-28, human, porcine, rat; vasoactive intestinalpeptide 11-28, human, porcine, rat, ovine; vasoactive intestinal peptide(cod, Gadus morhua); vasoactive intestinal peptide 6-28; vasoactiveintestinal peptide antagonist; vasoactive intestinal peptide antagonist({Ac-Tyr¹, D-Phe²}-GHRF 1-29 amide); vasoactive intestinal peptidereceptor antagonist (4-Cl-D-Phe⁶, Leu¹⁷}-VIP); and vasoactive intestinalpeptide receptor binding inhibitor, L-8-K. Additional constructs includebut are not limited to, Ala {^(11,22,28)}VIP, Ala{^(2,8,9,11,19,22,24,25,27,28)} VIP, {K¹⁵, R¹⁶, L²⁷}-VIP(1-7)/GRF(8-27),Ro25-1553, Ro25-1392, BAY55-9837, R3P65, Maxadilan, PG97-269, PG99-465,Max.d.4, and M65 (Dickson & Finlayson, Pharmacology & Therapeutics,Volume 121, Issue 3, March 2009, Pages 294-316).

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into an analog. In general, the introducednon-naturally encoded amino acids are substantially chemically inerttoward the 20 common, genetically-encoded amino acids (i.e., alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline). In some embodiments, the non-naturally encoded amino acidsinclude side chain functional groups that react efficiently andselectively with functional groups not found in the 20 common aminoacids (including but not limited to, azido, ketone, aldehyde andaminooxy groups) to form stable conjugates. For example, an analog thatincludes a non-naturally encoded amino acid containing an azidofunctional group can be reacted with a polymer (including but notlimited to, poly(ethylene glycol) or, alternatively, a secondpolypeptide containing an alkyne moiety to form a stable conjugateresulting for the selective reaction of the azide and the alkynefunctional groups to form a Huisgen {3+2} cycloaddition product.

In some embodiments, the composition or pharmaceutical compositions ofthe claimed invention comprises an analog of a polypeptide, wherein theanalog amino acid sequence is based upon the fragments, polypeptides,polymers and functional deriviatives disclosed herein or combinationsthereof and wherein the analog comprises at least one or a plurality ofnon-natural amino acids and at least one or a plurality of β-amino acidresidues. A non-natural amino acid typically possesses an R group thatis any substituent other than one component of the twenty natural aminoacids, and may be suitable for use in the present invention. Because thenon-naturally encoded amino acids of the invention typically differ fromthe natural amino acids only in the structure of the side chain, thenon-naturally encoded amino acids form amide bonds with other aminoacids, including but not limited to, natural or non-naturally encoded,in the same manner in which they are formed in naturally occurringpolypeptides. However, the non-natural amino acids have side chaingroups that distinguish them from the natural amino acids. For example,R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether,thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amino group, or the like or any combination thereof.Other non-naturally occurring amino acids of interest that may besuitable for use in the present invention include, but are not limitedto, amino acids comprising a photoactivatable cross-linker, spin-labeledamino acids, fluorescent amino acids, metal binding amino acids,metal-containing amino acids, radioactive amino acids, amino acids withnovel functional groups, amino acids that covalently or noncovalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, amino acids comprising biotin or a biotin analogue, glycosylatedamino acids such as a sugar substituted serine, other carbohydratemodified amino acids, keto-containing amino acids, amino acidscomprising polyethylene glycol or polyether, heavy atom substitutedamino acids, chemically cleavable and/or photocleavable amino acids,amino acids with an elongated side chains as compared to natural aminoacids, including but not limited to, polyethers or long chainhydrocarbons, including but not limited to, greater than about 5 orgreater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-natural amino acids that may be suitable for use in thepresent invention and that are useful for reactions with water solublepolymers include, but are not limited to, those with carbonyl, aminooxy,hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups.In some embodiments, non-naturally encoded amino acids comprise asaccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of skill in the art. In someembodiments, the invention relates to a method of manufacturing apolypeptide analog wherein the polypeptide analog is manufactured usinga synthesis technique disclosed in the following references, which areincorporated herein by reference: For organic synthesis techniques, see,e.g., Organic Chemistry by Fessendon and Fessendon, (1982, SecondEdition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistryby March (Third Edition, 1985, Wiley and Sons, New York); and AdvancedOrganic Chemistry by Carey and Sundberg (Third Edition, Parts A and B,1990, Plenum Press, New York). See, also, U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, which is incorporated byreference herein. In addition to unnatural (or non-natural) amino acidsthat contain novel side chains, unnatural amino acids that may besuitable for use in the present invention also optionally comprisemodified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III of U.S. PatentApplication Publication 2010-0048871, wherein Z typically comprises OH,NH₂, SH, NH—R′, or S—R; X and Y, which can be the same or different,typically comprise S or O, and R and R′, which are optionally the sameor different, are typically selected from the same list of constituentsfor the R group described above for the unnatural amino acids as well ashydrogen. For example, unnatural amino acids of the invention optionallycomprise substitutions in the amino or carboxyl group as illustrated byFormulas II and III. Unnatural amino acids of this type include, but arenot limited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β amino acids such as substituted β-alanine.

In some embodiments, the composition or pharmaceutical compositions ofthe claimed invention comprises an analog of a polypeptide, wherein theanalog amino acid sequence is based upon the fragments, polypeptides,and functional deriviatives disclosed herein and wherein the analogcomprises at least one or a plurality of unnatural amino acid ornon-natural amino acid and at least one or a plurality of β-amino acidresidues, wherein the unnatural amino acids based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like, and aresuitable for use in the present invention. Tyrosine analogs include, butare not limited to, para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, where the substitutedtyrosine comprises, including but not limited to, a keto group(including but not limited to, an acetyl group), a benzoyl group, anamino group, a hydrazine, an hydroxyamine, a thiol group, a carboxygroup, an isopropyl group, a methyl group, a C₆-C₂₀ straight chain orbranched hydrocarbon, a saturated or unsaturated hydrocarbon, anO-methyl group, a polyether group, a nitro group, an alkynyl group orthe like. In addition, multiply substituted aryl rings are alsocontemplated. Glutamine analogs that may be suitable for use in thepresent invention include, but are not limited to, α.-hydroxyderivatives, cyclic derivatives, and amide substituted glutaminederivatives. Example phenylalanine analogs that may be suitable for usein the present invention include, but are not limited to,para-substituted phenylalanines, ortho-substituted phenyalanines, andmeta-substituted phenylalanines, where the substituent comprises,including but not limited to, a hydroxy group, a methoxy group, a methylgroup, an allyl group, an aldehyde, an azido, an iodo, a bromo, a ketogroup (including but not limited to, an acetyl group), a benzoyl, analkynyl group, or the like. Specific examples of unnatural amino acidsthat may be suitable for use in the present invention include, but arenot limited to, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an0-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002). Incorporation of azidesinto recombinant proteins for chemoselective modification by theStaudinger ligation, PNAS 99:19-24, for additional methionine analogs.

The chemical moieties via unnatural amino acids that can be incorporatedinto analogs offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. In some embodiments, the compositionor pharmaceutical compositions of the claimed invention comprises ananalog of a polypeptide, wherein the analog amino acid sequence is basedupon the fragments, polypeptides, and functional deriviatives disclosedherein and wherein the analog comprises at least one or a plurality ofunnatural amino acid or non-natural amino acid and at least one or aplurality of β-amino acid residues, wherein the unnatural amino is aphotoreactive unnatural amino acid chosen from (including but notlimited to, amino acids with benzophenone and arylazides (including butnot limited to, phenylazide) side chains), for example, allow forefficient in vivo and in vitro photocrosslinking of protein. Examples ofphotoreactive unnatural amino acids include, but are not limited to,p-azido-phenylalanine and p-benzoyl-phenylalanine. The protein with thephotoreactive unnatural amino acids can then be crosslinked at will byexcitation of the photoreactive group-providing temporal control. In oneexample, the methyl group of an unnatural amino can be substituted withan isotopically labeled, including but not limited to, methyl group, asa probe of local structure and dynamics, including but not limited to,with the use of nuclear magnetic resonance and vibrational spectroscopy.Alkynyl or azido functional groups, for example, allow the selectivemodification of proteins with molecules through a {3+2} cycloadditionreaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a second reactivegroup different from the NH₂ group normally present in α-amino acids. Asimilar non-natural amino acid can be incorporated at the carboxylterminus with a second reactive group different from the COOH groupnormally present in α-amino acids.

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of skill in theart. For organic synthesis techniques, see, e.g., Organic Chemistry byFessendon and Fessendon, (1982, Second Edition, Willard Grant Press,Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985,Wiley and Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).Additional publications describing the synthesis of unnatural aminoacids include, e.g., WO 2002/085923 entitled “In vivo incorporation ofUnnatural Amino Acids;” Matsoukas et al., (1995) J. Med. Chem., 38,4660-4669; King, F. E. & Kidd, D. A. A. (1949) A New Synthesis ofGlutamine and of γ-Dipeptides of Glutamic Acid from PhthylatedIntermediates. J. Chem. Soc., 3315-3319; Friedman, 0. M. & Chattenji, R.(1959) Synthesis of Derivatives of Glutamine as Model Substrates forAnti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al.(1988) Absolute Configuration of the Enantiomers of7-Chloro-4{{4-(diethylamino)-}-methylbutyl}amino}quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, patent applications entitled “Protein Arrays,” filed Dec. 22,2003, Ser. No. 10/744,899 and Ser. No. 60/435,821 filed on Dec. 22,2002.

Any of the compositions above may be used in the methods disclosed inthis instant specification.

In another embodiment of the invention, the composition comprises a VIPanalog, wherein the analog does not comprise a repetitive pattern ofβ-amino acids from the amino-terminus to the carboxy-terminus selectedfrom the following: ααααααβ, αααααβα, ααααβαα, αααβααα, ααβαααα,αβααααα, βαααααα, αααααββ, ααααββα, αααββαα, ααββααα, αββαααα, ββααααα,βαααααβ, βααααβα, βαααβαα, βααβααα, βαβαααα, αβααααβ, αβαααβα, αβααβαα,αβαβααα, ααβαααβ, ααβααβα, ααβαβαα, αααβααβ, αααβαβα, and ααααβαβ.

Some embodiments of the claimed invention include pharmaceuticalcompositions. In some embodiments, the pharmaceutical compositioncomprises any of the aforementioned compositions in combination with apharmaceutically acceptable carrier. In another embodiment of theinvention, the pharmaceutical composition comprises a VIP analog andfused, covalently bound to or linked by chemical linkage to one otheractive agent. In some embodiments, the chemical linkage disclosed hereinis used.

In another embodiment of the invention, the pharmaceutical compositioncomprises a VIP analog and one other active agent, wherein the VIPanalog comprises at least one α-amino acid and at least one β-aminoacid.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog is either: (a) an agonist of VPAC2 receptor; or (b) interfereswith VPAC2 receptor signaling pathway and comprises the followingrepetitive pattern of β-amino acids from the amino-terminus to thecarboxy terminus: β₁α₁α₂β₂α₃α₄α₅β₃, wherein β₁, β₂ and β₃ are β-homoamino acids; wherein there is an optional β amino acid (β₀ or β₄) to oneof the β-homo amino acids (e.g. β₁₋₃); wherein the composition,pharmaceutical compositions, kit, or polypeptides of the inventionoptionally comprise amino acids with a pattern selected from: ααααααβ,αααααβα, ααααβαα, αααβααα, ααβαααα, αβααααα, βαααααα, αααααββ, ααααββα,αααββαα, ααββααα, αββαααα, ββααααα, βαααααβ, βααααβα, βαααβαα, βααβααα,βαβαααα, αβααααβ, αβαααβα, αβααβαα, αβαβααα, ααβαααβ, ααβααβα, ααβαβαα,αααβααβ, αααβαβα, and ααααβαβ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog does not comprises a repetitive pattern of sequential β-aminoacids from the amino-terminus to the carboxy-terminus chosen from thefollowing: βααβαααβααβαααβααα; βααβαααβααβαααββαα; βααβαααβααβαααβββα;and βααβαααβααβαααββββ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog that does not comprise a pattern of sequential α or β-amino acidsfrom the amino-terminus to the carboxy-terminus chosen from thefollowing: ββαβαααβααβαααβααβ; βαββαααβααβαααβααβ; βααββααβααβαααβααβ;βααβαβαβααβαααβααβ; βααβααββααβαααβααβ; βααβαααββαβαααβααβ;βααβαααβαββαααβααβ; βααβαααβααββααβααβ; βααβαααβααβαβαβααβ;βααβαααβααβααββααβ; βααβαααβααβαααββαβ; and βααβαααβααβαααβαββ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog does not comprise a repetitive pattern of sequential β-aminoacids from the amino-terminus to the carboxy-terminus chosen from thefollowing: ββααβααβαααβααβααα; βαβαβααβαααβααβααα; βααββααβαααβααβααα;βαααββαβαααβααβααα; βαααβαββαααβααβααα; βαααβααββααβααβααα;βαααβααβαβαβααβααα; βαααβααβααββααβααα; βαααβααβαααββαβααα;βαααβααβαααβαββααα; βαααβααβαααβααββαα; βαααβααβαααβααβαβα; andβαααβααβαααβααβααβ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog doe not comprise a repetitive pattern of sequential β-amino acidsfrom the amino-terminus to the carboxy-terminus chosen from thefollowing: βααβαααβααβαααβααα; βααβαααβααβαααββαα; βααβαααβααβαααβββα;and βααβαααβααβαααββββ; wherein any α-amino acid residue may be anon-natural amino acid.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog comprises a repetitive pattern of sequential β-amino acids fromthe amino-terminus to the carboxy-terminus chosen from the following:βααβαααβααβαααβαα; βααβαααβααβαααββαα; βααβαααβααβαααβββα; andβααβαααβααβαααββββ; wherein at least one α-amino acid residue may be anon-natural amino acid.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog comprises a repetitive pattern of sequential β-amino acids fromthe amino-terminus to the carboxy-terminus chosen from the following:βααβαααβααβαααβααα; βααβαααβααβαααββαα; βααβαααβααβαααβββα; andβααβαααβααβαααββββ; wherein from about 1 to about 10 α-amino acidresidues may be a non-natural amino acid.

In any of the above-mentioned patterns one or more of the β-amino acidresidues may be replaced or modified with cyclic β-amino acid(cyclically-constrained beta amino acid), such as APC or ACPC; whereinthe C-terminus is, optionally, amidated; and wherein the N-terminus is,optionally, acylated; or functional fragments thereof.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprises a VIP analog, comprisingFTENYTKLRK (Seq ID No. 228) wherein at least two adjacent amino acidsare replaced by two sequential β-amino acids. In some embodiments, thecomposition, pharmaceutical compositions, kits, or polypeptides of theinvention comprise a VIP analog, wherein the at least two adjacentβ-amino acids are x and y.

The invention further relates to uses of a composition comprising a VIPanalog in the preparation of a medicament for treating or preventingpulmonary hypertension, primary arterial hypertension, pulmonaryhypertension associated to post-ventricular septal defect, idiopathicpulmonary fibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small lung cellcancer, autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, hyperglycemia, diabetes, insulin resistance,metabolic acidosis, obesity, Type I diabetes, Type II diabetes MultipleSclerosis, osteoporosis, Sjogren's syndrome, pancreatitis,uveoretinitis, osteoporosis, female sexual dysfunction. The inventionfurther relates to use of a composition comprising a VIP analog in thepreparation of a medicament for treating or preventing pulmonaryhypertension, primary arterial hypertension, pulmonary hypertensionassociated to post-ventricular septal defect, idiopathic pulmonaryfibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small lung cellcancer, autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, hyperglycemia, diabetes, insulin resistance,metabolic acidosis, obesity, Type I diabetes, Type II diabetes MultipleSclerosis, osteoporosis, Sjogren's syndrome, pancreatitis,uveoretinitis, osteoporosis, female sexual dysfunction.

In some embodiments, the invention relates to methods of manufacturingany one of the aforementioned compositions, pharmaceutical compositions,or a pharmaceutical salt derived therefrom comprising catalyzing areaction between at least one α-amino acid with at least one β-aminoacid.

The invention also relates to methods of treating or preventingpulmonary hypertension, primary arterial hypertension, pulmonaryhypertension associated to post-ventricular septal defect, idiopathicpulmonary fibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small lung cellcancer, autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, hyperglycemia, diabetes, insulin resistance,metabolic acidosis, obesity, Type I diabetes, Type II diabetes MultipleSclerosis, osteoporosis, Sjogren's syndrome, pancreatitis,uveoretinitis, osteoporosis, female sexual dysfunction comprisingadministrating any one of the compositions or pharmaceuticalcompositions comprising a VIP family analog, or a pharmaceutical saltderived therefrom, to a subject in need thereof.

The present invention also relates to methods of selectively targetingtumor cells which over-express the VPAC1 receptor. Specific analogsdescribed herein are capable of high-affinity and high selectivitybinding to the VPAC1 receptor in agonist mode. This selective bindingenables a highly targeted approach to selective killing of tumor cellsthat over-express these receptors. By incorporating chemotherapeuticpayloads to these compounds, these cells can be specifically targetedfor chemotherapeutic delivery. This targeting ability greatly improvesthe side-effect profile of systemicily available chemotherapeutics bydelivering the toxic drug to tumor cells that express this receptor. Thepayloads cover a variety of biological mechanisms including tubulininhibitors, DNA intercalators to name a few. These payload molecules canbe covalently linked to the targeting parent molecules described hereinfor optional cleavage within the lysosomal endocytosis pathway as eithera function of pH or enzymatic release. The internalization process ispart of the endogenous molecular biology of this receptor uponactivation. Payloads can be either a single molecule or a combination(linear or otherwise) of known chemotherapeutic candidates. Payloadlinkages can be achieved using either polymer or amino acid basedapproaches. Examples of cleavable linkers include di-peptides such asPro-Pro or Cit-Val (Cit being citrulline). Linkages typically occur oneither free amine or cysteine groups. Linkage chemistry is typically aresult of click chemistry and can be conducted while the peptide analogsdescribed herein are still attached to solid support by using orthogonalprotecting groups resulting in site-specific protecting group removalthus enabling direct linkage at the desired position. It is alsopossible to link payloads on either the c or n termini of the peptideanalogs described herein. Covalently attached payloads may also containnon-functional spacers in order to properly position the payload for itsdesired action. These non-functional spacers could be a combination ofpolyethylene glycol or amino acid derivatives (α, β or gamma amino acidin nature). The length of these spacers fulfills the requiredbiophysical orientation between the targeting molecule and its toxicpayload.

The present invention also relates to methods of inhibiting secretion ofTNF-α in a subject comprising administering a composition comprising avasoactive intestinal peptide (VIP) analog to a subject, wherein saidanalog comprises at least one of the sequences disclosed herein. In someembodiments, the method comprises administering the compositioncomprising any of the percentages of β-amino acids disclosed herein.

The present invention is also directed towards kits comprising any ofthe aforementioned compositions or pharmaceutical compositionscomprising a VIP analog, wherein the VIP analog comprises an α-aminoacid and at least one β-amino. The present invention is directed towardkits comprising any of the aforementioned compositions or pharmaceuticalcompositions comprising a VIP analog, wherein the VIP analog comprisesan α-amino acid and at least on β-amino acid. In some embodiments, thekit further comprises a vehicle for administration of the composition.

The present invention also relates to methods of identifying a modulatorof VIP receptor activity comprising:

a) contacting a human receptor with a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the human receptor inthe presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the humanreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human receptor in the absence of anunknown compound.

The present invention also relates to methods of identifying a modulatorof animal receptor activity comprising:

a) contacting an animal receptor with a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the animal receptor inthe presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the animalreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the animal receptor in the absence ofan unknown compound.

In addition, imaging agents (Tc99 based or other well-known agents) maybe covalently or non-covalently attached to either the Cor N-termini orfree-amine groups of the analogs described herein. These agents enablethe molecular detection of cell-surface receptors using well-knowntechnologies (e.g. PET and MRI). The ability to detect cells thatoverexpress the VPAC1 receptor is a valuable diagnostic tool forclinical medicine. In addition, these imaging agents may be used incombination with either the optional spacers described above and/or thecytotoxic payloads also described earlier, thus resulting in atherapeutic agent with reporting capabilities. In some embodiments, theinvention relates to a method of imaging cellular receptors, comprisingadministering an analog described herein with a imaging agent. In someembodiments, the invention relates to a method of imaging cellularreceptors, comprising administering an analog described hereincovalently bound to an imaging agent. In some embodiments, the imaginingagent can be read by CAT scan, PET scan, or MRI.

The present invention also relates to methods of identifying a modulatorof human VIP receptor activity comprising:

a) contacting a human VIP receptor with a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the human VIP receptorin the presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the human VIPreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound.

The present invention also relates to methods of identifying a modulatorof human VIP receptor activity comprising:

a) contacting a human VIP receptor with the VIP analog, wherein theanalog comprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the human VIP receptorin the presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the human VIPreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a representative in-vitro protease stability of VPAC-2selective analog (LBT-V208) compared to the native VIP as well as theVPAC2 selective agonist Ro 25-1553. It is noteworthy that the stabilityis greater for the non-lactam containing LBT-V208 as compared to Ro25-1553, which contains one lactam bridge between lysine and asparticacid residues.

FIG. 2 depicts a representative dose response curve for ligand mediatedreceptor internalization, resulting in a relative chemiluminescentsignal for SeqID 22. EC50 value is 1.024e-008 Molar.

FIG. 3 depicts a representative in-vitro protease stability of VPAC-1selective analog (LBT-V101) compared to the native VIP.

FIG. 4 depicts a pharmacokinetic study of seqID: 208 via subcutaneousadministration in C57 BL/6 male mice. Analysis conducted via LC/MS/MStechniques to confirm presence of target molecule. Points represent AUCfor each time point described in the x axis

FIG. 5 depicts an adoptive transfer of splenocytes from drug-treateddonors is neuroprotective in vivo. A. Photomicrographs of TH+/Niss1+neurons in the SN and TH+ striatal termini in mice treated with PBS,MPTP, or MPTP followed by adoptive transfer of splenocytes from eitherVIP, VPAC1, or VPAC2-treated mice. Sections were immunostained withanti-TH and HRP-conjugated secondary Ab and visualized with DAB. SNsections were counterstained with thionin. B. Total number of survivingdopaminergic neurons (TH+/Niss1+) and nondopaminergic neurons(TH−/Niss1+) in the SN following MPTP treatment and adoptive transfer.Percentages of spared dopaminergic neurons are included for eachtreatment. C. Relative TH densitometry of total dopaminergic termini inthe striatum. B and C. Differences in means (±SEM, n=8 mice per group)were determined where P<0.05 compared with groups treated with PBS (a)and MPTP (b). VIP, VPAC1 (seq id 101) and VPAC2 (seq id 1189) armsreceived 15 ug/day/5 days.

FIG. 6 depicts an average Mac-1+ expression for each of the arm of thestudy. Mice were administered 15 ug VIP, VPAC1 (Seq id 101), VPAC2 (seqid 208) per day for 5 days prior to MPTP challenge. Mice were sacrificedand Mac-1+ levels were determined 2 days later.

FIG. 7 depicts an adoptive transfer of splenocytes from drug-treateddonors is neuroprotective in vivo. Total number of survivingdopaminergic neurons (TH+/Niss1+) and nondopaminergic neurons(TH−/Niss1+) in the SN following direct administration and MPTPtreatment. Percentages of spared dopaminergic neurons are included foreach treatment. Differences in means (±SEM, n=8 mice per group) weredetermined where P<0.05 compared with groups treated with PBS (a) andMPTP (b). VIP, VPAC1 (seq id 101) and VPAC2 (seq id 208) arms received15 ug/day/5 days.

FIG. 8 depicts an inhibition assay to assess the suppressive function ofTreg isolated from each donor group which includes VPAC1 (Seq ID 101),VPAC2 (Seq ID 1189), VIP and PBS (no treatment) on proliferation ofanti-CD3 stimulated naïve CD4⁺CD25⁻ T cells. injections given daily/5days/i.p., VIP 15 ug, VPAC1 and VPAC2 300 ug. To assess whether VIP,VPAC1, 2 and PBS splenocytes (SPC) suppress effector T cellproliferative responses, we evaluated SPC co-cultures from VIP-treateddonors for their proliferative capacity in the presence of eitheranti-CD3. At a 1:1 ratio of SPC to VIP SPC, proliferation to anti-CD3stimulation were suppressed by xx % and yy %, respectively anddiminished in a dose dependent fashion with the diminution of VIP SPCnumber. We hypothesized that Treg function stimulated by VIP, VPAC 1,2.To test this hypothesis, we evaluated CD4⁺CD25⁺CD62L^(low) Treg isolatedfrom naïve and VIP-treated mice for their capacity to inhibitCD3-mediated proliferation of CD4⁺CD25⁻ naïve T cells. VIP Treg showedincreased functional capacity to suppress T cell proliferation comparedwith nave Treg showing a consistent or greater inhibition ofproliferation. VIP and VPAC 1,2 Treg showed enhanced suppressivecapacity compared to PBS Treg populations, with YY % greater inhibitionversus naïve Treg at a 1:1 Treg to responder ratio. These data suggestedthat VPAC 1, 2 enhances T cell regulatory function.

FIG. 9 depicts an IP Glucose Tolerance Test for VPAC1 (Seq ID: 1131) andVPAC2 (seq id: 1189) agonists. DIO mice were fasted overnight followedby subcutaneous delivery of the specific VIP agonist. 60 minutes later,a bolus injection of glucose was delivered intraperitoneally. Glucosereading were taken at the following timepoints −60, 0, 15, 45, 60, 90and 120 minutes post glucose delivery.

FIG. 10 depicts NMR spectra of TMC (in DCl) prepared from chitosan withviscosity of 5 mPa. The large peak observed at 3.65 is assigned to thetri-methylated state of chitosan.

FIG. 11 depicts Formula I.

FIG. 12 depicts Formula II.

FIG. 13 depicts Formula III.

FIG. 14 depicts Formula IV.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

The term “active state” refers to the conformation or set ofconformations of a polypeptide that allows functional domain or domainsof the polypeptide to associate or disassociate with another compound,macromolecule, or ligand. In some embodiments, the association ordisassociation of the polypeptide with another compound, macromolecule,or ligand may propagate or inhibit a biologic signal.

The terms “amino acid” refer to a molecule containing both an aminogroup and a carboxyl group bound to a carbon which is designated theα-carbon. Suitable amino acids include, without limitation, both the D-and L-isomers of the naturally-occurring amino acids, as well asnon-naturally occurring amino acids prepared by organic synthesis orother metabolic routes. In some embodiments, a single “amino acid” mighthave multiple sidechain moieties, as available per an extended aliphaticor aromatic backbone scaffold. Unless the context specifically indicatesotherwise, the term amino acid, as used herein, is intended to includeamino acid analogs.

The term “analog” refers to any polypeptide comprising at least oneα-amino acid and at least one β-amino acid residue, wherein thepolypeptide is structurally similar to a naturally occurring full-lengthprotein and shares the biochemical or biological activity of thenaturally occurring full-length protein upon which the analog is based.In some embodiments, an analog is any polypeptide comprising at leasttwo contiguous β-amino acid residues, wherein the polypeptide isstructurally similar to a naturally occurring full-length protein andshares the biochemical or biological activity of the naturally occurringfull-length protein upon which the analog is based and wherein theaddition of one or more β-amino acid residues constrains an alphahelical structure in the polypeptide. In some embodiments, an analog isany polypeptide comprising at least three contiguous β-amino acidresidues, wherein the polypeptide is structurally similar to a naturallyoccurring full-length protein and shares the biochemical or biologicalactivity of the naturally occurring full-length protein upon which theanalog is based.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term analog includes polypeptidesconjugated to a polymer such as PEG and may be comprised of one or moreadditional derivitizations of cysteine, lysine, or other residues. Inaddition, analogs of the instant invention may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid, or may be conjugated to anaturally encoded amino acid utilizing techniques known in the art suchas coupling to lysine or cysteine.

Polymer modification of polypeptides has been reported. U.S. Pat. No.4,904,584 discloses PEGylated lysine depleted polypeptides, wherein atleast one lysine residue has been deleted or replaced with any otheramino acid residue. WO 99/67291 discloses a process for conjugating aprotein with PEG, wherein at least one amino acid residue on the proteinis deleted and the protein is contacted with PEG under conditionssufficient to achieve conjugation to the protein. WO 99/03887 disclosesPEGylated variants of polypeptides belonging to the growth hormonesuperfamily, wherein a cysteine residue has been substituted with anon-essential amino acid residue located in a specified region of thepolypeptide. WO 00/26354 discloses a method of producing a glycosylatedpolypeptide variant with reduced allergenicity, which as compared to acorresponding parent polypeptide comprises at least one additionalglycosylation site. U.S. Pat. No. 5,218,092 discloses modification ofgranulocyte colony stimulating factor (G-CSF) and other polypeptides soas to introduce at least one additional carbohydrate chain as comparedto the native polypeptide. Examples of PEGylated peptides includeGW395058, a PEGylated peptide thrombopoietin receptor (TPOr) agonist (deSerres M., et al., Stem Cells. 1999; 17(4):203-9), and a PEGylatedanalogue of growth hormone releasing factor (PEG-GRP; D'Antonio M, etal. Growth Horm IGF Res. 2004 June; 14(3):226-34).

The term analog also includes glycosylated analogs, such as but notlimited to, analogs glycosylated at any amino acid position, N-linked orO-linked glycosylated forms of the polypeptide. In addition, splicevariants are also included. The term analog also includes heterodimers,homodimers, heteromultimers, or homomultimers of any one or morepolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogs containing, for example, specific deletions or othermodifications yet maintain biological activity.

In some embodiments, the non-natural amino acid residue is a monomer ofan aliphatic polypeptide. In some embodiments the aliphatic analogs arechosen from oligoureas, azapeptides, pyrrolinones, α-aminoxy-peptides,and sugar-based peptides. In some embodiments, the composition comprisesa non-natural β-amino acid. In some embodiments, the analog or activeagent couple thereto is a fragment of the full-length protein ormacromolecule. In some embodiments, fragments are from about 5 to about500 amino acids in length as compared to the naturally occurring, fullytranslated and fully processed protein sequences. In some embodiments,the analogs comprise a fragment of a naturally translated full-lengthprotein that induces the biochemical or biological activity of abiological pathway of a subject at a level equivalent to or increased ascompared to the activity induced by a naturally occurring full-lengthprotein upon which the analog is derived. In some embodiments, theanalog is a truncated polypeptide as compared to the full-length,naturally translated or naturally occurring polypeptide upon which thetruncated polypeptide is derived. In some embodiments, the analog is asynthetic polypeptide, wherein at least one of the amino acid residuesof the polypeptide comprises at least one non-natural side chain. Insome embodiments, the analogs of the invention comprise at least onenon-natural amino acid chosen from one of the following structures:aminoisobutyric acid, 3-Aminobutyric acid, and2-hydroxy-4-(4-nitrophenyl)butyric acid. In some embodiments, the analoghas a polypeptide backbone of identical length and similar homology tothe polypeptides disclosed in Table 1. In some embodiments, the analogis about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homolgous to at leastone of the polypeptides disclosed in Tables 1. In some embodiments, theanalog is an agonist or antagonist of one or more of the followingreceptors: VPAC1, VPAC2, or PAC1. In some embodiments the analog of thepresent invention is modified by a bioactive lipid moiety on at leastone amino acid residue of the analog. In such embodiments, the lipidmoieties may be chosen from the following lipid molecules: LPA,progesterone, prostanoids, SIP, LPA, cannabinoids,2-arachidonylglycerol.

TABLE 1 Identifier Sequence Seq ID LBT-V201 Ac-HSDAVFTENYTKLRKQLA x KKYx ND l KKG g T (Seq ID 1) LBT-V202 Ac-HSDAVFTENYTKLRKQLAA z KY x NDL kKG g T (Seq ID 2) LBT-V203 Ac-HSDAVFTENYTKLRKQ x AA z KYL x DL k KGGT(Seq ID 3) LBT-V204 Ac-HBDAVFTENYTKLRKQLAA z KY x NDL k KG g T (Seq ID4) LBT-V205 Ac-HBDA v FTENYTKLRKQLAA z KY x NDL k KG g T (Seq ID 5)LBT-V206 Ac-HBDA v FTE n YTKLRKQLAA z KY x NDL k KG g T (Seq ID 6)LBT-V207 Ac-H x DAVFTENYTKLRKQLAA z KY x NDL k KG g T (Seq ID 7)LBT-V208 Ac-H x DA x FTENYTKLRKQLAA z KY x NDL k KG g T (Seq ID 8)LBT-V209 Ac-H x DA x FTE x YTKLRKQLAA z KY x NDL k KG g T (Seq ID 9)LBT-V210 Ac-H x DA v FTENYTKLRKQLAA z KY x NDL k KG g T (Seq ID 10)LBT-V211 Ac-H x DA v FTENYTKLRKQ l AA z KY x NDL k KG g T (Seq ID 11)LBT-V212 Ac-H x DA v FTENYTKLRK q LAA z KY x NDL k KG g T (Seq ID 12)LBT-V213 Ac-H x DA v FTENYTKLR k QLAA z KY x NDL k KG g T (Seq ID 13)LBT-V214 Ac-H x DA v FTENYTKL r KQLAA z KY x NDL k KG g T (Seq ID 14)LBT-V215 Ac-H x DA v FTENYTK l RKQLAA z KY x NDL k KG g T (Seq ID 15)LBT-V216 Ac-H x DA x FTE ny TKLRKQLAA z KY x NDL k KG g T (Seq ID 16)LBT-V217 Ac-H x DA x FTE xy TKLRKQ l AA z KY x NDL k KG g T (Seq ID 17)LBT-V218 Ac-H x DA x FTE xy TKLRK g LAA z KY x NDL k KG g T (Seq ID 18)LBT-V219 Ac-H x DA x FTE xy TKLR k QLAA z KY x NDL k KG g T (Seq ID 19)LBT-V220 Ac-HSDAVFTENYTKLRKQ x AAK z YL x DLK k GGT (Seq ID 20) LBT-V221Ac-HSDAVFTDNYTRLRKQ x AAK z YL x SIK n KRY (Seq ID 21) LBT-V101HSDAVFTDNY t RLR k QLA v KKY l NAI l N (Seq ID 22) LBT-V102 H x DAVFTDNYt RLR k QLA v KKY l NAI l N (Seq ID 23) LBT-V103 H x DA x FTDNY t RLR kQLA v KKY l NAI l N (Seq ID 24) LBT-V104 H x DA v FTDNY t RLR k QLA vKKY l NAI l N (Seq ID 25) LBT-V105 H s DA v FTDNY t RLR k QLA v KKY lNAI l N (Seq ID 26) LBT-V106 H s DA v FTD n Y t RLR k QLA v KKY l NAI lN (Seq ID 27) LBT-V107 HS d AVF t DNY t RLR k QLA v KKY l NAI l N (SeqID 28) LBT-V108 HS d AVFTDNY t RLR k QLA v KKY l NAI l N (Seq ID 29)LBT-V109 HSD a VFTDNY t RLR k QLA v KKY l NAI l N (Seq ID 30) LBT-V110HSDAV f TDNY t RLR k QLA v KKY l NAI l N (Seq ID 31) LBT-V111 HSDAVF tDNY t RLR k QLA v KKY l NAI l N (Seq ID 32) LBT-V112 HSDAVFT d NY t RLRk QLA v KKY l NAI l N (Seq ID 33) LBT-V113 HSDAVFTD n Y t RLR k QLA vKKY l NAI l N (Seq ID 34) LBT-V114 HSDAVFTDNY yt RLR k QLA v KKY l NAI lN (Seq ID 35) LBT-V115 HSDAV f TDNY yt RLR k QLA v KKY l NAI l N (Seq ID36) LBT-V116 HSDAVF t DNY t RLR k QLA v KKY l NAI l N (Seq ID 37)LBT-V117 HSD a VF t DNY t RLR k QLA v KKY l NAI l N (Seq ID 38) LBT-V118HSDAVFTNSY r KVLkRLS a RKL l QDI l (Seq ID 39) LBT-V119 HSDAVFTNSYR kVLK r LSA r KLL q DIL (Seq ID 40) LBT-V120 HSDAVFTNS y RKV l KRL s ARK lLQD i L (Seq ID 41) LBT-V121 H x DA x FTNSY r KVL k RLS a RKL l QDI l(Seq ID 42) LBT-V122 H x DA x FTN xy RKVLK r LSA z KL x QDI l (Seq ID43) LBT-V123 HSDAVFTNSYRKVLK r LSA r KL l QDI l (Seq ID 44) LBT-V124HSDAVFTNSYRKVLK r LS a RKL l QD i L (Seq ID 45) LBT-V125HSDAVFTNSYRKVLKR l SA r KLL q DI l (Seq ID 46) LBT-V126 HfDAVFTDNY t RLRk QLA v KKY l NAI l N (Seq ID 47) LBT-V127 HfDA x FTDNY t RLR k QLA vKKY l NAI l N (Seq ID 48) LBT-V128 HfDA v FTDNY t RLR k QLA v KKY

NAI l N (Seq ID 49) LBT-V129 HfDA v FTD n Y t RLR k QLA v KKY l NAI l N(Seq ID 50) LBT-V130 Hf d AVF t DNY t RLR k QLA v KKY l NAI l N (Seq ID51) LBT-V131 HfDAVFTNSY r KVL k RLS a RKL l QDI l (Seq ID 52) LBT-V132HfDAVFTNSYR k VLK r LSA r KLL q DIL (Seq ID 53) LBT-V133 HfDAVFTNS y RKVl KRL s ARK l LQD i L (Seq ID 54) LBT-V134 HfDA x FTNSY r KVL k RLS aRKL l QDI l (Seq ID 55) LBT-V135 HfDA x FTN xy RKVLK r LSA z KL x QDI l(Seq ID 56) LBT-V136 HfDAVFTNSYRKVLK r LSA r KL l QDI l (Seq ID 57)LBT-V137 HfDAVFTNSYRKVLK r LS a RKL l QD i L (Seq ID 58) LBT-V138HfDAVFTNSYRKVLKR l SA r KLLqDI l (Seq ID 59) LBT-V222 Ac-H x DA x FTE xyTKLRK q LAA z KY x NDL k K g GT (Seq ID 60) LBT-V223 Ac-H x DA x FTE xyTKLRK q LAA z KY x NDL k K gg T (Seq ID 61) LBT-V224 Ac-H x DA x FTE xyTKLRK q LAA z KY x NDL k KGG t (Seq ID 62) LBT-V225 Ac-H x DA x FTE xyTKLRK q LAA z KY x NDL k K g G t (Seq ID 63) LBT-V226 Ac-H x DA x FTE xyTKLRK q LAA z KY x NDL k KG gt (Seq ID 64) LBT V201-LBT-V226 are VPAC₂selective LBT V101-LBT-V138 are VPAC₁ selective Lowercase, bold andunderlined represent Homo-Beta3 amino acids Italic, lowercase Frepresents d-Phenylalanine Lowercase, bold and underlined x representsAPCP (2-aminocyclopentane carboxylic acid)

The term “α-amino acid” refers to any and all natural and unnaturalα-amino acids and their respective residues (i.e., the form of the aminoacid when incorporated into a polypeptide molecule), without limitation.In some embodiments, “α-amino acid” explicitly encompasses theconventional and well-known naturally occurring amino acids, as well asall synthetic variations, derivatives, and analogs thereof. In someembodiments, “α-amino acid” means alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and/or valine. In some embodiments,α-amino acids also include analogs such as N-methylated α-amino acids,hydroxylated α-amino acids, and aminoxy acids. In some embodiments,α-amino refers to include N-alkyl α-amino acids (such as N-methylglycine), hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, nor-valine,nor-leucine, and ornithine.

The terms “β-amino acid” and “β-amino acid residue” refer to any and allβ-amino acids and their respective residues (i.e., the form of the aminoacid when incorporated into a polypeptide molecule), without limitation.In some embodiments, the terms “β-amino acid” refers to those β-aminoacids described in U.S. Pat. No. 6,060,585, issued May 9, 2000,incorporated herein by reference, and those described in allowed U.S.Pat. No. 6,683,154, issued Jan. 27, 2004; U.S. Pat. No. 6,710,186,issued Mar. 23, 2004; and U.S. Pat. No. 6,727,368, issued Apr. 27, 2004,all of which are incorporated herein by reference. Further still, cyclicimino carboxylic acids and gem-di-substituted cyclic imino carboxylicacids (both of which are a type of cyclically-constrained β-amino acid)may also be used in the invention. In some embodiments, the term“β-amino acid” refers to residues disclosed in U.S. Pat. No. 6,958,384,issued Oct. 25, 2005, incorporated herein by reference. Further still,these β-residues may also take the form of the gem-di-substituted cyclicimino acids disclosed in U.S. Pat. No. 6,710,186, incorporated herein byreference. In some embodiments, the terms “β-amino acid” refers toβ-homo amino acids. In some embodiments the β-amino acids refers to theselection of an amino acid chosen from the following:

R¹ is selected from the group consisting hydrogen and an aminoprotecting group; R² is selected from the group consisting of hydrogenand a carboxy protecting group; and when R³ is bonded to a carbon atom,R³ is selected from the group consisting of hydrogen, hydroxy, linear orbranched C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl, mono-or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O,and S; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl, —(CH₂)_(n+1), —OR⁴, —(CH₂)_(n+1)—SR⁴,—(CH₂)_(n+1)—S(═O)—CH₂—R⁴, —(CH₂)_(n+1)—S(═O)₂—CH₂—R⁴,—(CH₂)_(n+1)—NR⁴R⁴, —(CH₂)_(n+1)—NHC(═O)R⁴,—(CH₂)_(n+1)—NHS(═O)₂—CH₂—R⁴, —(CH₂)_(n+1)—O—(CH₂)_(m)—R⁵,—(CH₂)_(n+1)—S—(CH₂)_(n+1)R⁵, —(CH₂)_(n+1)—S(═O)—(CH₂)_(m)—R⁵,—(CH₂)_(n+1)—S(═O)₂—(CH₂)_(m)—R⁵, —(CH₂)_(n+1)—NH—(CH₂)_(m)—R⁵,—(CH₂)_(n+1)—N—{(CH₂)_(m)—R⁵}₂, —(CH₂)_(n+1)—NHC(═O)—(CH₂)_(n+1)—R⁵, and—(CH₂)_(n+1)—NH S(═O)₂—(CH₂)_(m)—R⁵; wherein each R⁴ is independentlyselected from the group consisting of hydrogen, C₁-C₆alkyl, alkenyl, oralkynyl; mono- or bicyclic aryl, mono- or bicyclic heteroaryl having upto S heteroatoms selected from N, O, and S; mono- or bicyclicaryl-C₁-C₆alkyl, mono- or bicyclic heteroaryl-C₁-C₆alkyl; and wherein R⁵is selected from the group consisting of hydroxy, C₁-C₆alkyloxy,aryloxy, heteroaryloxy, thio, C₁-C₆alkylthio, C₁-C₆alkylsulfinyl,C₁-C₆alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl,heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- ordi-C₁-C₆alkylamino, mono- or diarylamino, mono- or diheteroarylamino,N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,N-aryl-N-heteroarylamino, aryl-C₁-C₆alkylamino, carboxylic acid,carboxamide, mono- or di-C₁-C₆alkylcarboxamide, mono- ordiarylcarboxamide, mono- or diheteroarylcarboxamide,N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide,N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- ordi-C₁-C₆alkylsulfonamide, mono- or diarylsulfonamide, mono- ordiheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea;mono- di- or tri-substituted urea, wherein the subsitutent(s) isselected from the group consisting of C₁-C₆alkyl, aryl, heteroaryl;O-alkylurethane, O-arylurethane, and O-heteroarylurethane; and m is aninteger of from 2-6 and n is an integer of from 0-6; and when R³ isbonded to a nitrogen atom, R³ is independently selected from the groupconsisting of those listed above for when R³ is attached to a carbonatom, and further selected from the group consisting of —S(═O)₂—CH₂—R⁴,—C(═O)—R⁴—S(═O)₂—(CH₂)_(m)R⁵, and —C(═O)—(CH₂)_(n+1)—R⁵; wherein R⁴ andR⁵ are as defined hereinabove, and m is an integer of from 2-6 and n isan integer of from 0-6; provided that when the β-amino acid is offormula R³ is not hydrogen; racemic mixtures thereof, isolated orenriched enantiomers thereof; isolated or enriched diastereomers thereofand salts thereof. In some embodiments the β-amino acids refers to theselection of an amino acid chosen from the following:

In some embodiments the β-amino acids refers to the following Formula I(FIG. 11):

In some embodiments the β-amino acids refers to the following Formula II(FIG. 12):

wherein the NH₂ and/or COOH groups are replaced with functional peptidebonds.

In some embodiments the term “β-amino acid” refers to the followingFormula III (FIG. 13):

-   -   wherein X and Y combined, together with the carbon atoms to        which they are bonded, define a substituted or unsubstituted        C₄-C₈ cycloalkyl, cycloalkenyl or heterocyclic ring having one        or more nitrogen atoms as the sole heteroatom;    -   the substituents on carbon atoms of the rings being        independently selected from the group consisting of linear or        branched C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic        aryl, mono- or bicyclic heteroaryl having up to 5 heteroatoms        selected from N, O, and S; mono- or bicyclic aryl-C₁-C₆-alkyl,        mono- or bicyclic heteroaryl-C₁-C₆alkyl, —(CH₂)_(n+1)—OR⁴,        —(CH₂)_(n+1)—SR⁴, —(CH₂)_(n+1)—S(═O)—CH₂—R⁴,        —(CH₂)_(n+1)—S(═O)₂—CH₂—R⁴, —(CH₂)_(n+1)—NR⁴R⁴,        —(CH₂)_(n+1)—NHC(═O)R⁴, —(CH₂)_(n+1)—NHS(═O)₂—CH₂—R⁴,        —(CH₂)_(n+1)—O—(CH₂)_(m)—R⁵, —(CH₂)_(n+1)—S—(CH₂)_(m)—R⁵,        —(CH₂)_(n+1)—S(═O)—(CH₂)_(m)—R⁵,        —(CH₂)_(n+1)—S(═O)₂—(CH₂)_(m)—R⁵, —(CH₂)_(n+1)—NH—(CH₂)_(m)—R⁵,        —(CH₂)_(n+1)—N—{(CH₂)_(m)—R⁵}₂,        —(CH₂)_(n+1)—NHC(═O)—(CH₂)_(n+1)—R⁵, and        —(CH₂)_(n+1)—NHS(═O)₂—(CH₂)_(m)—R⁵;    -   wherein R⁴ is independently selected from the group consisting        of hydrogen, C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic        aryl, mono- or bicyclic heteroaryl having up to 5 heteroatoms        selected from N, O, and S; mono- or bicyclic aryl-C₂-C₆-alkyl,        mono- or bicyclic heteroaryl-C₁-C₆-alkyl; and    -   wherein R⁵ is selected from the group consisting of hydroxy,        C₃-C₆-alkyloxy, aryloxy, heteroaryloxy, thio, C₁-C₆-alkylthio,        C₁-C₆-alkylsulfinyl, C₁-C₆-alkylsulfonyl, arylthio,        arylsulfinyl, arylsulfonyl, heteroarylthio, heteroarylsulfinyl,        heteroarylsulfonyl, amino, mono- or di-C₁-C₆-alkylamino, mono-        or diarylamino, mono- or diheteroarylamino, N-alkyl-N-arylamino,        N-alkyl-N-heteroarylamino, N-aryl-N-heteroarylamino,        aryl-C₁-C₆-alkylamino, carboxylic acid, carboxamide, mono- or        di-C₂-C₆-alkylcarboxamide, mono- or diarylcarboxamide, mono- or        diheteroarylcarboxamide, N-alkyl-N-arylcarboxamide,        N-alkyl-N-heteroarylcarboxamide, N-aryl-N-heteroarylcarboxamide,        sulfonic acid, sulfonamide, mono- or di-C₂-C₈-alkylsulfonamide,        mono- or diarylsulfonamide, mono- or diheteroarylsulfonamide,        N-alkyl-N-arylsulfonamide, N-alkyl-N-heteroarylsulfonamide,        N-aryl-N-heteroarylsulfonamide, urea; mono- di- or        tri-substituted urea, wherein the subsitutent(s) is selected        from the group consisting of C₁-C₆-alkyl, aryl, heteroaryl;        O-alkylurethane, O-arylurethane, and O-heteroarylurethane; and    -   m is in integer of from 2-6 and n is an integer of from 0-6;    -   the substituents on heteroatoms of the ring being independently        selected from the group consisting of        —S(═O)²—CH₂—R⁴—C(═O)—R⁴—S(═O)₂—(CH₂)_(m)—R⁵, and        —C(═O)—(CH₂)_(n+1)—R⁵; wherein R⁴ and R⁵ are as defined        hereinabove, and m is an integer of from 2-6 and n is an integer        of from 0-6;    -   provided that when X & Y together with the carbons to which they        are bonded define a five- or six-membered cycloalkyl or a        live-membered heterocyclic ring having one nitrogen as the sole        heteroatom, and the nitrogen is bonded to a carbon atom adjacent        to the carboxy carbon of Formula I, the cycloalkyl or        heterocyclic ring is substituted;    -   R¹ is selected from the group consisting hydrogen and an amino        protecting group;    -   R² is selected from the group consisting of hydrogen and a        carboxy protecting group;    -   racemic mixtures thereof, isolated or enriched enantiomers        thereof; isolated or enriched diastereomers thereof;    -   and salts thereof.

In some embodiments the term “β-amino acid” refers to selection of anamino acid chosen from the following: β³ or β². In some embodiments theterm “β-amino acid” refers to selection of an amino acid chosen from thefollowing Formula IV (FIG. 14):

wherein R, R′, R″, and R′″ are any substituent.

In some embodiments the term “β-amino acid” refers to selection of anamino acid chosen from the following Formula IV (FIG. 14):

wherein R, R′, R″, and R′″ is an amine, hydroxy, hydroxyl, carbonyl, H,═O, —OH, —COOH, —N, —CH₃, —CH₂—X, halo, aryl, arylalkoxy, arylalkyl,alkynyl, alkenyl, alkylene, alkyl, akyl-halo, arylamido,alkylheterocycle, alkylamino, alkylguanidino, alkanol, alkylcarboxy,cycloalkyl, heteroaryl, heteroarylalkyl, heteroarylalkoxy, orheterocyclyl;wherein X is any substituent.

In some embodiments the term “β-amino acid” refers to selection of anamino acid chosen from the following Formula IV (FIG. 14):

wherein R, R′, R″, and R′″ are any substituent, provided that: (i) R isnot O, N, or halo when the R is in a β³-residue, (ii) R and R′ are notO, N, or halo when the R and R′ are in a β^(3,3)-residue; (iii) R is notO, N, or halo when the R is in a β^(2,3)-residue; (iv) R and R′ are notO, N, or halo when the R and R′ are in a β^(2,3,3)-residue; (v) R″ isnot O, N, or halo when the R″ is in a β^(2,2,3)-residue; (vi) R and R′are not O, N, or halo when the R and R′ are in a β^(2,2,3,3)-residue.

In some embodiments the term “β-amino acid” refers to selection of anamino acid chosen from the following:

wherein R, R′, R″, and R′″ is an amine, hydroxy, hydroxyl, carbonyl, H,═O, —OH, —COOH, —N, —CH₃, —CH₂—X, halo, aryl, arylalkoxy, arylalkyl,alkynyl, alkenyl, alkylene, alkyl, akyl-halo, arylamido,alkylheterocycle, alkylamino, alkylguanidino, alkanol, alkylcarboxy,cycloalkyl, heteroaryl, heteroarylalkyl, heteroarylalkoxy, orheterocyclyl;wherein X is any substituent; provided that: (i) R is not O, N, or halowhen the R is in a β³-residue, (ii) R and R′ are not O, N, or halo whenthe R and R′ are in a β^(3,3)-residue; (iii) R is not O, N, or halo whenthe R is in a β^(2,3)-residue; (iv) R and R′ are not O, N, or halo whenthe R and R′ are in a β^(2,3,3)-residue; (v) R″ is not O, N, or halowhen the R″ is in a β^(2,2,3)-residue; (vi) R and R′ are not O, N, orhalo when the R and R′ are in a β^(2,2,3,3)-residue.A “cyclic” beta-amino acid is acid is an amino acid of the followingformula I:

wherein X and Y combined, together with the carbon atoms to which theyare bonded, define a substituted or unsubstituted C₄-C₈ cycloalkyl orcycloalkenyl group; wherein substituents on carbon atoms of the ringsbeing independently selected from the group consisting of linear orbranched C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl, mono-or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O,and S; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl, —(CH2)_(n+1)—OR₄, —(CH2)_(n+1)—SR₄,—(CH₂)_(n+1)—S(═O)—CH₂—R₄, —(CH₂)_(n+1)—S(═O)₂—CH₂—R⁴,—(CH₂)_(n+1)—NR₄R₄, —(CH₂)_(n+1)—NHC(═O)R₄,—(CH₂)_(n+1)—NHS(═O)₂—CH₂—R₄, —(CH₂)_(n+1)—O—(CH₂)_(m)—R₅,—(CH₂)_(n+1)—S—(CH₂)_(m)—R₅, —(CH₂)_(n+1)—S(═O)—(CH₂)_(m)—R₅,—(CH₂)_(n+1)—S(═O)₂—(CH₂)_(m)—R₅, —(CH₂)_(n+1)—NH—(CH₂)_(m)—R₅,—(CH₂)_(n+1)—N—{(CH₂)_(m)—R₅}₂, —(CH₂)_(n+1)—NHC(═O)—(CH₂)_(n+1)—R₅, and—(CH₂)_(n+1)—NHS(═O)₂—(CH₂)_(m)—R₅; wherein R₄ is independently selectedfrom the group consisting of hydrogen, C₁-C₆-alkyl, alkenyl, or alkynyl;mono- or bicyclic aryl, mono- or bicyclic heteroaryl having up to 5heteroatoms selected from N, O, and S; mono- or bicyclicaryl-C₁-C₆-alkyl, mono- or bicyclic heteroaryl-C₁-C₆-alkyl; and whereinR₅ is selected from the group consisting of hydroxy, C₁-C₆-alkyloxy,aryloxy, heteroaryloxy, thio, C₁-C₆-alkylthio, C₁-C₆-alkylsulfinyl,C₁-C₆-alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl,heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- ordi-C₁-C₆-alkylamino, mono- or diarylamino, mono- or diheteroarylamino,N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,N-aryl-N-heteroarylamino, aryl-C₁-C₆-alkylamino, carboxylic acid,carboxamide, mono- or di-C₁-C₆-alkylcarboxamide, mono- ordiarylcarboxamide, mono- or diheteroarylcarboxamide,N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide,N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- ordi-C₁-C₆-alkylsulfonamide, mono- or diarylsulfonamide, mono- ordiheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea;mono- di- or tri-substituted urea, wherein the subsitutent(s) isselected from the group consisting of C₁-C₆-alkyl, aryl, heteroaryl;O-alkylurethane, O-arylurethane, and O-heteroarylurethane; and m is aninteger of from 2-6 and n is an integer of from 0-6; the substituents onheteroatoms of the ring being independently selected from the groupconsisting of —S(═O)₂—CH₂—R₄—C(═O)—R₄—S(═O)₂—(CH₂)_(m)—R₅, and—C(═O)—(CH₂)_(n+1)—R₅; wherein R₄ and R₅ are as defined hereinabove, andm is an integer of from 2-6 and n is an integer between 0 and 6;provided that when X and Y together with the carbons to which they arebonded define a five- or six-membered cycloalkyl or a five-memberedheterocyclic ring having one nitrogen as the sole heteroatom, and thenitrogen is bonded to a carbon atom adjacent to the carboxy carbon ofFormula I, the cycloalkyl or heterocyclic ring is substituted; R₁ isselected from the group consisting hydrogen and an amino protectinggroup; R₂ is selected from the group consisting of hydrogen and acarboxy protecting group; racemic mixtures thereof, isolated or enrichedenantiomers thereof; isolated or enriched diastereomers thereof; andsalts thereof.

A “heterocyclic” beta-amino acid is an amino acid of formula I, whereinX and Y combined, together with the carbon atoms to which they arebonded, define a substituted or unsubstituted C₄-C₈ cyclically orcycloalkenyl group having one or more nitrogen, oxygen or sulfur atomsas a heteroatom(s) within the cycloakyl or cycloalkenyl group; whereinsubstituents on carbon atoms of the cycloakyl or cycloalkenyl ringsbeing independently selected from the group consisting of linear orbranched C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl, mono-or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O,and S; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl, —(CH2)_(n+1)—OR₄, —(CH2)_(n+1)—SR₄,—(CH₂)_(n+1)—S(═O)—CH₂—R₄, —(CH₂)_(n+1)—S(═O)₂—CH₂—R₄,—(CH₂)_(n+1)—NR₄R₄, —(CH₂)_(n+1)—NHC(═O)R₄,—(CH₂)_(n+1)—NHS(═O)₂—CH₂—R₄, —(CH₂)_(n+1)—O—(CH₂)_(m)—R₅,—(CH₂)_(n+1)—S—(CH₂)_(m)—R₅, —(CH₂)_(n+1)—S(═O)—(CH₂)_(m)—R₅,—(CH₂)_(n+1)—S(═O)₂—(CH₂)_(m)—R₅, —(CH₂)_(n+1)—NH(CH₂)_(m)—R₅,—(CH₂)_(n+1)—N—{(CH₂)_(m)—R₅}₂, —(CH₂)_(n+1)—NHC(═O)—(CH₂)_(n+1)—R₅, and—(CH₂)_(n+1)—NHS(═O)₂—(CH₂)_(m)—R₅; wherein R₄ is independently selectedfrom the group consisting of hydrogen, C₁-C₆-alkyl, alkenyl, or alkynyl;mono- or bicyclic aryl, mono- or bicyclic heteroaryl having up to 5heteroatoms selected from N, O, and S; mono- or bicyclicaryl-C₁-C₆-alkyl, mono- or bicyclic heteroaryl-C₁-C₆-alkyl; and whereinR₅ is selected from the group consisting of hydroxy, C₁-C₆-alkyloxy,aryloxy, heteroaryloxy, thio, C₁-C₆-alkylthio, C₁-C₆-alkylsulfinyl,C₁-C₆-alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl,heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- ordi-C₁-C₆-alkylamino, mono- or diarylamino, mono- or diheteroarylamino,N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,N-aryl-N-heteroarylamino, aryl-C₁-C₆-alkylamino, carboxylic acid,carboxamide, mono- or di-C₁-C₆-alkylcarboxamide, mono- ordiarylcarboxamide, mono- or diheteroarylcarboxamide,N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide,N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- ordi-C₁-C₆-alkylsulfonamide, mono- or diarylsulfonamide, mono- ordiheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea;mono- di- or tri-substituted urea, wherein the subsitutent(s) isselected from the group consisting of C₁-C₆-alkyl, aryl, heteroaryl;O-alkylurethane, O-arylurethane, and O-heteroarylurethane; and m is aninteger of from 2-6 and n is an integer of from 0-6; the substituents onheteroatoms of the ring being independently selected from the groupconsisting of —S(═O)₂—CH₂—R₄—C(═O)—R₄—S(═O)₂—(CH₂)_(m)—R₅, and—C(═O)—(CH₂)_(n+1)—R₅; wherein R₄ and R₅ are as defined hereinabove, andm is an integer of from 2-6 and n is an integer between 0 and 6;provided that when X and Y together with the carbons to which they arebonded define a five- or six-membered cycloalkyl or a five-memberedheterocyclic ring having one nitrogen as the sole heteroatom, and thenitrogen is bonded to a carbon atom adjacent to the carboxy carbon ofFormula I, the cycloalkyl or heterocyclic ring is substituted; R₁ isselected from the group consisting hydrogen and an amino protectinggroup; R₂ is selected from the group consisting of hydrogen and acarboxy protecting group; racemic mixtures thereof, isolated or enrichedenantiomers thereof; isolated or enriched diastereomers thereof; andsalts thereof.

In some embodiments, at least one of the β-amino acid residues in theanalog is replaced with at least one β-amino acid residue that iscyclically constrained via a ring encompassing its iv and β³ carbonatoms. In another embodiment of the invention, most or all of theinserted β-amino acid residues are cyclically constrained. In anotherversion of the invention, at least one of the β-amino acid residues isunsubstituted at its iv and β³ carbon atoms. Alternatively, all of theβ-amino acid residues may be substituted at their β² and β³ carbon atoms(with linear, branched or cyclic substituents). In some embodiments, thecyclic substituents of the claimed invention comprise side chains thatare covalently bonded to the side chains of other contiguous aminoacids. In some embodiments, the cyclic substituents of the claimedinvention comprise side chains that are covalently bonded to the sidechains of other non-contiguous amino acids. In some embodiments thecyclic substituents of the claimed invention do not include side chainsthat are covalently bonded to the side chains of other contiguous ornon-contiguous amino acids. In some embodiments the terms beta-3 orbeta-2 amino acid refers to β3-homo β2-homo amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C, H), nonpolar side chains(e.g., G, A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T,V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a VIP analog, for example, replacedwith another amino acid residue from the same side chain family. Otherexamples of acceptable substitutions are substitutions based onisosteric considerations (e.g. norleucine for methionine) or otherproperties (e.g. 2-thienylalanine for phenylalanine).

As used herein, the term “derived from” in the context of therelationship between a chemical structure or amino acid sequence and arelated chemical structure or related amino acid sequence describes achemical structure or amino acid sequence that may be homologous to orstructurally similar to the related chemical structure or related aminoacid sequence.

As used herein, the term “inflammatory disease” refers to any disease,condition, or ailment that results from an immune response or a pathogeninfection, which in some instances may be characterized by one or moreof pain, swelling, and redness of a tissue types. In some embodiments,inflammatory disease refers to rheumatoid arthritis, Crohn's disease,sepsis, ulcerative colitis, irritable bowel disease, chronic irritablebowel syndrome, and allergies such as allergic rhinitis.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., a short domain ofVIP) without abolishing or substantially altering its essentialbiological or biochemical activity (e.g., receptor binding oractivation). An “essential” amino acid residue is a residue that, whenaltered from the wild-type sequence of the polypeptide, results inabolishing or substantially abolishing the polypeptide's essentialbiological or biochemical activity.

A “non-natural side chain” is a modified or synthetic chain of atomsjoined by covalent bond to the α-carbon atom, β-carbon atom, or γ-carbonatom which does not make up the backbone of the polypeptide chain ofamino acids. The natural side chain, or R group, of alanine is a methylgroup. In some embodiments, the non-natural side chain of thecomposition is a methyl group in which on e or more of the hydrogenatoms is replaced by a deuterium atom.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full-lengthproteins (e.g., fully processed pro-proteins or full-length syntheticpolypeptides) as well as shorter amino acid sequences (e.g., fragmentsof naturally-occurring proteins or synthetic polypeptide fragments).

The term “salt” refers to acidic salts formed with inorganic and/ororganic acids, as well as basic salts formed with inorganic and/ororganic bases. Examples of these acids and bases are well known to thoseof ordinary skill in the art. Such acid addition salts will normally bepharmaceutically acceptable although salts of non-pharmaceuticallyacceptable acids may be of utility in the preparation and purificationof the compound in question. Salts include those formed fromhydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric,lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic andbenzenesulphonic acids.

In some embodiments, salts of the compositions comprising either a VIPor VIP analog may be formed by reacting the free base, or a salt,enantiomer or racemate thereof, with one or more equivalents of theappropriate acid. In some embodiments, pharmaceutical acceptable saltsof the present invention refer to analogs having at least one basicgroup or at least one basic radical. In some embodiments, pharmaceuticalacceptable salts of the present invention comprise a free amino group, afree guanidino group, a pyrazinyl radical, or a pyridyl radical thatforms acid addition salts. In some embodiments, the pharmaceuticalacceptable salts of the present invention refer to analogs that are acidaddition salts of the subject compounds with (for example) inorganicacids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, orwith suitable organic carboxylic or sulfonic acids, for examplealiphatic mono- or di-carboxylic acids, such as trifluoroacetic acid,acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acidor oxalic acid, or amino acids such as arginine or lysine, aromaticcarboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid,2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid,aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamicacid, heteroaromatic carboxylic acids, such as nicotinic acid orisonicotinic acid, aliphatic sulfonic acids, such as methane-, ethane-or 2-hydroxyethane-sulfonic acid, or aromatic sulfonic acids, forexample benzene-, p-toluene- or naphthalene-2-sulfonic acid. Whenseveral basic groups are present mono- or poly-acid addition salts maybe formed. The reaction may be carried out in a solvent or medium inwhich the salt is insoluble or in a solvent in which the salt issoluble, for example, water, dioxane, ethanol, tetrahydrofuran ordiethyl ether, or a mixture of solvents, which may be removed in vacuoor by freeze drying. The reaction may also be a metathetical process orit may be carried out on an ion exchange resin. In some embodiments, thesalts may be those that are physiologically tolerated by a patient.Salts according to the present invention may be found in their anhydrousform or as in hydrated crystalline form (i.e., complexed or crystallizedwith one or more molecules of water).

The term “subject” is used throughout the specification to describe ananimal to whom treatment with the compositions according to the presentinvention is provided or administered. For treatment of those conditionswhich are specific for a specific subject, such as a human being, theterm “patient” may be interchangeably used. In some instances in thedescription of the present invention, the term “patient” will refer tohuman patients. In some embodiments, the subject may be a mammal to whomthe present invention is provided or administered. In some embodiments,the subject may be a non-human animal to whom the present invention isprovided or administered.

The term “soluble” or “water soluble” refers to solubility that ishigher than 1/100,000 (mg/ml). The solubility of a substance, or solute,is the maximum mass of that substance that can be dissolved completelyin a specified mass of the solvent, such as water. “Practicallyinsoluble” or “insoluble,” on the other hand, refers to an aqueoussolubility that is 1/10,000 (mg/ml) or less. Water soluble or solublesubstances include, for example, polyethylene glycol. In someembodiments, the polypeptide of the claimed invention may be bound bypolyethylene glycol to better solubilize the composition comprising thepeptide.

The terms “treating” and “to treat”, mean to alleviate symptoms,eliminate the causation either on a temporary or permanent basis, or toprevent or slow the appearance of symptoms. The term “treatment”includes alleviation, elimination of causation (temporary or permanent)of, or prevention of symptoms and disorders associated with anycondition. The treatment may be a pre-treatment as well as a treatmentat the onset of symptoms.

“Effective amount” refers to an amount of a compound, material, orcomposition, as described herein effective to achieve a particularbiological result such as, but not limited to, biological resultsdisclosed, described, or exemplified herein. Such results may include,but are not limited to, the effective reduction of symptoms associatedwith any of the disease states mentioned herein, as determined by anymeans suitable in the art. The effective amount of the composition maybe dependent on any number of variables, including without limitation,the species, breed, size, height, weight, age, overall health of thesubject, the type of formulation, the mode or manner or administration,the type and/or severity of the particular condition being treated, orthe need to modulate the activity of the molecular pathway induced byassociation of the analog to its receptor. The appropriate effectiveamount can be routinely determined by those of skill in the art usingroutine optimization techniques and the skilled and informed judgment ofthe practitioner and other factors evident to those skilled in the art.A therapeutically effective dose of the analogs described herein mayprovide partial or complete biological activity as compared to thebiological activity induced by the wild-type or naturally occurringpolypeptides upon which the analogs are derived. A therapeuticallyeffective dose of the analogs described herein may provide a sustainedbiochemical or biological affect and/or an increased resistance todegradation when placed in solution as compared with the normal affectobserved when the naturally occurring and fully processed translatedprotein is administered to the same subject.

The term “fragment” refers to any analog of a naturally occurringpolypeptide disclosed herein that comprises at least 4 amino acidsidentical to the naturally occurring polypeptide upon which the analogis based. The term “functional fragment” refers to any fragment of anyanalog of a naturally occurring polypeptide disclosed herein thatcomprises at least 4 amino acids identical to the naturally occurringpolypeptide upon which the analog is based and shares the function ofthe naturally occurring polypeptide upon which the analog is based. Insome embodiments, the compositions or pharmaceutical compositioncomprises an analog comprising at least one β-amino acid. wherein theanalog is a fragment of VIP, a VIP family member, an interleukin, or anyof the polypeptides disclosed in the instant application. In someembodiments, the compositions or pharmaceutical composition comprises ananalog comprising at least one β-amino acid, wherein the analog is afragment of VIP, a VIP family member, an interleukin, or any of thepolypeptides disclosed in the instant application and wherein thefragment shares at least 4 contiguous amino acid residues with thenaturally occurring polypeptide upon which the analog is based andwherein the fragment retains the biological activity of the naturallyoccurring polypeptide upon which the analog is based. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 26 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 25 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 24 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 23 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 22 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 21 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 20 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 19 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 18 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 19 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 17 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 16 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 15 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 14 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about β amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 12 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 11 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 10 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment that comprises between about 1to about 9 amino acids of the naturally occurring VIP sequence. In someembodiments, the VIP analog is a fragment of VIP that comprises betweenabout 1 to about 8 amino acids of the naturally occurring VIP sequence.In some embodiments, the VIP analog is a fragment that comprises betweenabout 1 to about 7 amino acids of the naturally occurring VIP sequence.In some embodiments, the VIP analog is a fragment that comprises betweenabout 1 to about 6 amino acids of the naturally occurring VIP sequence.In some embodiments, the VIP analog is a fragment that comprises betweenabout 1 to about 5 amino acids of the naturally occurring VIP sequence.In some embodiments, the VIP analog is a fragment that comprises betweenabout 1 to about 4 amino acids of the naturally occurring VIP sequence.In some embodiments, the analog is modified with at least one PEGmolecule on at least one of the non-natural amino acids.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it. In some embodiments the alkyl group is chosen from: C₁-C₁₀,C₂-C₁₀, C₃-C₁₀, C₄-C₁₀, C₅-C₁₀, C₆-C₁₀, C₇-C₁₀, C₈-C₁₀, C₉-C₁₀, C₁-C₁₀,C₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₁-C₆, C₁-C₇, C₁-C₈, or C₁-C₉,

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having about 2 to about 20 (inclusive) carbonatoms in it.

The term “aryl” refers to an aromatic ring system. In some embodiments,the aryl group of the analog include substituents, wherein 0, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 atoms of each ring are substituted by asubstituent. In some embodiments, the aryl group refers to a 6-carbonmonocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2,3, or 4 atoms of each ring are substituted by a substituent. Examples ofaryl groups include phenyl, naphthyl and the like. The term “arylalkyl”or the term “aralkyl” refers to alkyl substituted with an aryl. The term“arylalkoxy” refers to an alkoxy substituted with aryl. “Arylalkyl”refers to an aryl group, as defined above, wherein one of the arylgroup's hydrogen atoms has been replaced with an alkyl group, as definedabove. Representative examples of an arylalkyl group include, but arenot limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl,2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl,3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl,4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl,2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH₂-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl.

“Alkylheterocycle” refers to an alkyl group, as defined above, whereinone of the alkyl group's hydrogen atoms has been replaced with aheterocycle. Representative examples of an alkylheterocyclo groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to an alkyl group, as defined above, wherein one ofthe alkyl group's hydrogen atoms has been replaced with a —C(O)NH₂group. Representative examples of an alkylamido group include, but arenot limited to, —CH₂C(O)NH₂, —CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂C(O)NH₂,—CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH(C(O)NH₂)CH₃,—CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃, —C(CH₃)₂CH₂C(O)NH₂,—CH₂CH₂NHC(O)CH₃, —CH₂CH₂NHC(O)CH₂CH₃, and —CH₂CH₂NHC(O)CH═CH₂.

“Alkylamino” refers to an alkyl group, as defined above, wherein one ofthe alkyl group's hydrogen atoms has been replaced with a —NH₂ group.Representative examples of an alkylamido group include, but are notlimited to —CH₂NH₂, CH₂CH₂NH₂, CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂.

“Alkylguanidino” refers to an alkyl group, as defined above, wherein oneof the alkyl group's hydrogen atoms has been replaced with a—NH₂(C═NH)NH₂ group. Representative examples of an alkylamido groupinclude, but are not limited to —CH₂NH₂(C═NH)NH₂, CH₂CH₂NH₂(C═NH)NH₂,CH₂CH₂CH₂NH₂(C═NH)NH₂, —CH₂CH₂CH₂CH₂NH₂(C═NH)NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂(C═NH)NH₂. In some embodiments alkyl units can befound on the N atom(s) of the alkylamino or alkylguanidino groups (forexample, —CH₂NH(CH₃), CH₂N(CH₃)₂).

“Alkanol” refers to an alkyl group, as defined above, wherein one of thealkyl group's hydrogen atoms has been replaced with a hydroxyl group.Representative examples of an alkanol group include, but are not limitedto, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to an alkyl group, as defined above, wherein oneof the alkyl group's hydrogen atoms has been replaced with a —COOHgroup. Representative examples of an alkylcarboxy group include, but arenot limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,3 to 8 carbons, or 3 to 6 carbons, wherein the cycloalkyl groupadditionally is optionally substituted. Some cycloalkyl groups include,without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-10 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include, but are not limited to, pyridyl, furyl orfuranyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,quinolinyl, indolyl, thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include, but are not limited to, piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the composition comprises an analog comprises oneor more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. Preparation of pure enantiomers or mixtures ofdesired enantiomeric excess (ee) or enantiomeric purity are accomplishedby one or more of the many methods of (a) separation or resolution ofenantiomers, or (b) enantioselective synthesis known to those of skillin the art, or a combination thereof. These resolution methods generallyrely on chiral recognition and include, for example, chromatographyusing chiral stationary phases, enantioselective host-guestcomplexation, resolution or synthesis using chiral auxiliaries,enantioselective synthesis, enzymatic and nonenzymatic kineticresolution, or spontaneous enantioselective crystallization. Suchmethods are disclosed generally in Chiral Separation Techniques: APractical Approach (2nd Ed.), G. Subramanian (ed.), Wiley-VCH, 2000; T.E. Beesley and R. P. W. Scott, Chiral Chromatography, John Wiley & Sons,1999; and Satinder Ahuja, Chiral Separations by Chromatography, Am.Chem. Soc., 2000. Furthermore, there are equally well-known methods forthe quantitation of enantiomeric excess or purity, for example, GC,HPLC, CE, or NMR, and assignment of absolute configuration andconformation, for example, CD ORD, X-ray crystallography, or NMR.

All tautomeric forms and isomeric forms and mixtures, whether individualgeometric isomers or stereoisomers or racemic or non-racemic mixtures,of a chemical structure or wntire analog is intended, unless thespecific stereochemistry or isomeric form is specifically indicated inthe analog name, chemical name or structure. All such isomeric forms ofthese compositions are included in the present invention unlessexpressly provided otherwise. In some embodiments, the analogs of thisinvention are also represented in multiple tautomeric forms, in suchinstances, the invention includes all tautomeric forms of the analogsdescribed herein (e.g., if alkylation of a ring system results inalkylation at multiple sites, the invention includes all such reactionproducts). All such isomeric forms of such analogs are included in thepresent invention unless expressly provided otherwise. All crystal formsof the analogs described herein are included in the present inventionunless expressly provided otherwise. All deuterated form of the analogsdescribed herein are included in the present invention. In someembodiments as least one hydrogen atom of the analog is replace with adeuterium atom. In some embodiments at least one hydrogen atom that isinvolved with a hydrogen-bond is replaced with a deuterium atom. In someembodiments at least one solvent exchangeable hydrogen atom is replacedwith a deuterium atom. In some embodiments, the compositions,pharmaceutical compositions, and analogs contained therein comprise fromabout 1% to about 100% of their hydrogen replaced with deuterium atoms.In some embodiments, the compositions, pharmaceutical compositions, andanalogs contained therein comprise from about 90% to about 100% of theirhydrogen replaced with deuterium atoms. In some embodiments, thecompositions, pharmaceutical compositions, and analogs contained thereincomprise from about 80% to about 90% of their hydrogen replaced withdeuterium atoms. In some embodiments, the compositions, pharmaceuticalcompositions, and analogs contained therein comprise from about 70% toabout 80% of their hydrogen replaced with deuterium atoms. In someembodiments, the compositions, pharmaceutical compositions, and analogscontained therein comprise from about 60% to about 70% of their hydrogenreplaced with deuterium atoms. In some embodiments, the compositions,pharmaceutical compositions, and analogs contained therein comprise fromabout 50% to about 60% of their hydrogen replaced with deuterium atoms.In some embodiments, the compositions, pharmaceutical compositions, andanalogs contained therein comprise from about 40% to about 50% of theirhydrogen replaced with deuterium atoms. In some embodiments, thecompositions, pharmaceutical compositions, and analogs contained thereincomprise from about 30% to about 40% of their hydrogen replaced withdeuterium atoms. In some embodiments, the compositions, pharmaceuticalcompositions, and analogs contained therein comprise from about 20% toabout 30% of their hydrogen replaced with deuterium atoms. In someembodiments, the compositions, pharmaceutical compositions, and analogscontained therein comprise from about 10% to about 20% of their hydrogenreplaced with deuterium atoms. In some embodiments, the compositions,pharmaceutical compositions, and analogs contained therein comprise fromabout 5% to about 10% of their hydrogen replaced with deuterium atoms.If the analog of the claimed invention includes a methyl group, adeutrated analog may have one, two, or three of the hydrogens replacedby deuterium atoms. In some embodiments, the analog may contain one ormore radioisotopes. In some embodiments, as least one hydrogen atom ofthe analog is replace with a tritium atom. In some embodiments, thecompositions, pharmaceutical compositions, and analogs contained thereincomprise from about 1% to about 5% of their hydrogens are replaced withtritium atoms.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.15) increase ordecrease of at least 1%, 2%, or 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, 10⁻¹², 10⁻¹¹, 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷,10⁻⁶, 10⁻⁵, 10⁻⁴ or any other real values ≧0 and ≦2 if the variable isinherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “biological activity” encompasses structural and functionalproperties of a macrocycle of the invention. Biological activity is, forexample, structural stability, alpha-helicity, affinity for a target(for example as measured by EC₅₀ or IC₅₀ values), resistance toproteolytic degradation, cell penetrability, intracellular stability, invivo stability, or any combination thereof.

The terms “prodrug” or “prodrug derivative” mean a covalently-bondedderivative or carrier of the analog of the claimed invention or activedrug substance which undergoes at least some biotransformation prior toexhibiting its pharmacological effect(s). In general, such prodrugs havemetabolically cleavable groups and are rapidly transformed in vivo toyield the analog of the claimed invention, for example, by hydrolysis inblood, and generally include esters and amide analogs of the analogs.The prodrug is formulated with the objectives of improved chemicalstability, improved patient acceptance and compliance, improvedbioavailability, prolonged duration of action, improved organselectivity, improved formulation (e.g., increased hydrosolubility),and/or decreased side effects (e.g., toxicity). In general, prodrugsthemselves have weak or no biological activity and are stable underordinary conditions. Prodrugs can be readily prepared from the analogsusing methods known in the art, such as those described in A Textbook ofDrug Design and Development, Krogsgaard-Larsen and H. Bundgaard (eds.),Gordon & Breach, 1991, particularly Chapter 5: “Design and Applicationsof Prodrugs”; Design of Prodrugs, H. Bundgaard (ed.), Elsevier, 1985;Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan (ed.), MarcelDekker, 1998; Methods in Enzymology, K. Widder et al. (eds.), Vol. 42,Academic Press, 1985, particularly pp. 309-396; Burger's MedicinalChemistry and Drug Discovery, 5th Ed., M. Wolff (ed.), John Wiley &Sons, 1995, particularly Vol. 1 and pp. 172-178 and pp. 949-982;Pro-Drugs as Novel Delivery Systems, T. Higuchi and V. Stella (eds.),Am. Chem. Soc., 1975; and Bioreversible Carriers in Drug Design, E. B.Roche (ed.), Elsevier, 1987, each of which is incorporated herein byreference in their entireties. In some embodiments, the analog may be aprodrug that, when administered to the subject becomes biologicallyactive.

In some embodiments, the invention relates to a composition orpharmaceutical composition comprising a pharmaceutically acceptableprodrug that, when administered to the subject becomes biologicallyactive. The term “pharmaceutically acceptable prodrug” as used hereinmeans a prodrug of a compound of the invention which is, within thescope of sound medical judgment, suitable for use in contact with thetissues of humans and lower animals without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible.

In some embodiments, the analog of the claimed invention is apharmaceutically-acceptable acid addition salt. The term“pharmaceutically-acceptable acid addition salt” means those salts whichretain the biological effectiveness and properties of the free bases andwhich are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, andthe like, and organic acids such as acetic acid, trichloroacetic acid,trifluoroacetic acid, adipic acid, alginic acid, ascorbic acid, asparticacid, benzenesulfonic acid, benzoic acid, 2-acetoxybenzoic acid, butyricacid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid,digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid,glycerophosphoric acid, hemisulfic acid, heptanoic acid, hexanoic acid,formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionicacid), lactic acid, maleic acid, hydroxymaleic acid, malic acid, malonicacid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid,naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid,oxalic acid, pamoic acid, pectinic acid, phenylacetic acid,3-phenylpropionic acid, picric acid, pivalic acid, propionic acid,pyruvic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid,sulfanilic acid, tartaric acid, p-toluenesulfonic acid, undecanoic acid,and the like. In some embodiments, the analog of the claimed inventionis a pharmaceutically-acceptable base addition salt. The term“pharmaceutically-acceptable base addition salt” means those salts whichretain the biological effectiveness and properties of the free acids andwhich are not biologically or otherwise undesirable, formed withinorganic bases such as ammonia or hydroxide, carbonate, or bicarbonateof ammonium or a metal cation such as sodium, potassium, lithium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike. Suitable salts include the ammonium, potassium, sodium, calcium,and magnesium salts. Salts derived from pharmaceutically-acceptableorganic nontoxic bases include salts of primary, secondary, and tertiaryamines, quaternary amine compounds, substituted amines includingnaturally occurring substituted amines, cyclic amines and basicion-exchange resins, such as methylamine, dimethylamine, trimethylamine,ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine,tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, tetramethylammonium compounds, tetraethylammoniumcompounds, pyridine, N,N-dimethylaniline, N-methylpiperidine,N-methylmorpholine, dicyclohexylamine, dibenzylamine,N,N-dibenzylphenethylamine, 1-ephenamine, N,N′-dibenzylethylenediamine,polyamine resins, and the like. In some embodiments, the composition ofthe claimed invention comprises at least one organic nontoxic baseschosen from isopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexylamine, choline, and caffeine.

The term “solvate” means a physical association of a compound with oneor more solvent molecules or a complex of variable stoichiometry formedby a solute (the analog of the claimed invention) and a solvent, forexample, water, ethanol, or acetic acid. This physical association mayinvolve varying degrees of ionic and covalent bonding, includinghydrogen bonding. In certain instances, the solvate will be capable ofisolation, for example, when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. Ingeneral, the solvents selected do not interfere with the biologicalactivity of the solute. Solvates encompasses both solution-phase andisolatable solvates. Representative solvates include hydrates,ethanolates, and methanolates.

The invention relates to compositions comprising an analog of anaturally occurring polypeptide sequence. In some embodiments theinvention relates to a composition comprising an analog of a naturallyoccurring polypeptide sequence wherein the analog is from about 80% to99% homologous to a naturally occurring polypeptide sequence. In someembodiments the invention relates to a composition comprising an analogof a naturally occurring polypeptide sequence wherein the analog is fromabout 80% to 85% homologous to a naturally occurring polypeptidesequence. In some embodiments the invention relates to a compositioncomprising an analog of a naturally occurring polypeptide sequencewherein the analog is from about 85% to 90% homologous to a naturallyoccurring polypeptide sequence. In some embodiments the inventionrelates to a composition comprising an analog of a naturally occurringpolypeptide sequence wherein the analog is from about 90% to 95%homologous to a naturally occurring polypeptide sequence. In someembodiments the invention relates to a composition comprising an analogof a naturally occurring polypeptide sequence wherein the analog is fromabout 95% to 99% homologous to a naturally occurring polypeptidesequence. In some embodiments the invention relates to a compositioncomprising an analog of a naturally occurring polypeptide sequencewherein the analog is about 95%, 96%, 97%, 98%, or 99% homologous to anaturally occurring polypeptide sequence. In some embodiments the analogis derived from the naturally occurring polypeptide of the VIP family.In some embodiments, the analog is derived from the naturally occurringpolypeptide of the VIP family and has at least one β-amino acid residueand/or at least one modified amino acid residue comprising APC or ACPC.VIP is

HSDGTFTSELSRLREGARLQRLLQGLV HSDAVFTDNYTRLRKQMAVKKYLNSILN

In some embodiments, the VIP analog is selective for one particularreceptor versus another. In some embodiments, the composition comprisesa VIP analog wherein the VIP analog is selective for, or preferentiallybinds to, VPAC1, VPAC2, PAC1, VIPR1, or VIPR2. In some embodiments, thecomposition comprises a VIP analog wherein the VIP analog is selectivefor, or preferentially binds, VPAC1. In some embodiments, thecomposition comprises a VIP analog wherein the VIP analog is selectivefor, or preferentially binds, VPAC2. In some embodiments, thecomposition comprises a VIP analog wherein the VIP analog is selectivefor, or preferentially binds, PAC1. In some embodiments, the compositioncomprises a VIP analog wherein the VIP analog is selective for, orpreferentially binds, VIPR1. In some embodiments, the compositioncomprises a VIP analog wherein the VIP analog is selective for, orpreferentially binds, VIPR2. In some embodiments, the VIP analog is anagonist of at least one of the following: VPAC1, VPAC2, PAC1, VIPR1, orVIPR2. In some embodiments, the VIP analog is an antagonist of at leastone of the following: VPAC1, PAC1, VIPR1, or VIPR2.

The invention relates to the manufacturing of a synthetic polypeptidewhich is an amino acid sequence that corresponds to the sequence of thepolypeptides disclosed herein or fragment thereof. In some embodiments,the synthetic polypeptides comprises a repeated pattern of αααβ but doesnot comprise any other repeated pattern of any alpha or beta aminoacids. In some embodiments, the invention relates to an animal cellcomprising any of the polypeptides disclosed herein. In someembodiments, animal cells can be contacted with the syntheticpolypeptide to induce the biochemical pathway or biological activityordinarily induced by the naturally occurring polypeptide upon which theanalog is based.

The compositions of the invention may be prepared by the syntheticchemical procedures described herein, as well as other proceduressimilar to those which may be used for making β-amino acid peptides.Such procedures include both solution and solid phase procedures, e.g.,using either Boc and Fmoc methodologies. The compounds of the inventionmay be synthesized using solid phase synthesis techniques.Fmoc-N-Protected β-amino acids can be used to synthesizepoly-α/β-peptides by conventional manual solid-phase synthesisprocedures under standard conditions on any number of solid supports,including ortho-chloro-trityl chloride resin. Esterification ofFmoc-β-amino acids with the ortho-chloro-trityl resin can be performedaccording to the method of Barbs et. al., Tetrahedron Lett., 1989, 30,3943. The resin (150 mg, 1.05 mmol Cl) is swelled in 2 ml CH₂Cl₂ for 10min. A solution of the Fmoc-protected β-amino acid in CH₂Cl₂ and iPr₂EtNare then added successively and the suspension is mixed under argon for4 h. Subsequently, the resin is filtered and washed withCH₂Cl₂/MeOH/iPr₂EtN (17:2:1, 3×3 min), CH₂Cl₂ (3×3 min), DMF (2×3 min),CH₂Cl₂ (3×3 min), and MeOH (2×3 min) The substitution of the resin isdetermined on a 3 mg sample by measuring the absorbance of thedibenzofulvene adduct at 300 nm. The Fmoc group is removed using 20%piperidine in DMF (4 ml, 2×20 min) under Ar bubbling. The resin is thenfiltered and washed with DMF (6×3 min) For each coupling step, asolution of the β-amino acid (3 equiv.), BOP (3 equiv.) and HOBT (3equiv.) in DMF (2 ml) and iPr₂EtN (9 eq) are added successively to theresin and the suspension is mixed for 1 h under Ar. Monitoring of thecoupling reaction is performed with 2,4,6-trinitrobenzene-sulfonic acid(TNBS) (W. S. Hancock and J. E. Battersby, Anal. Biochem. (1976), 71,260). In the case of a positive TNBS test (indicating incompletecoupling), the suspension is allowed to react for a further 1 h. Theresin is then filtered and washed with DMF (3×3 min) prior to thefollowing Fmoc deprotection step. After the removal of the last Fmocprotecting group, the resin is washed with DMF (6×3 min), CH₂Cl₂ (3×3min), Et₂O (3×3 min) and dried under vacuum for 3 h. Finally thepeptides are cleaved from the resin using 2% TFA in CH₂Cl₂ (2 ml, 5×15min) under Ar. The solvent is removed and the oily residues aretriturated in ether to give the crude α-/β-polypeptides. The compoundsare further purified by HPLC.

The compositions of the invention may be prepared by the syntheticchemical procedures described herein, as well as other proceduressimilar to those which may be used for making β-amino acid peptides.Such procedures include both solution and solid phase procedures, e.g.,using either Boc or Fmoc methodologies. The compounds of the inventionmay be synthesized using solid phase synthesis techniques.Fmoc-N-Protected β-amino acids can be used to synthesizepoly-α/β-peptides by conventional manual solid-phase synthesisprocedures under standard conditions on any number of solid supports,including ortho-chloro-trityl chloride resin, Wang resin (NovaBiochem0.75 mmol substitution) and Rink amid resin (NovaBiochem 0.55 mmolsubstitution). Resin is typically swelled in 100% DMF for 30 minutesthen deprotected using 20% piperidine in DMF for 2 minutes at 80° (3×).Fmoc protected amino acids (natural or non-natural) can then be coupledto the resin using a cocktail of AA:HATU:DIEA:Resin (3:2.5:4:1, LiCL0.8M final concentration) in DMF for 2 minutes at 70° (3×). The resin isthen washed (3×) with DMF, DCM (dichloromethane) (3×) and again with DMF(3×) between deprotection and coupling steps. Monitoring of the couplingreaction is performed with 2,4,6-trinitrobenzene-sulfonic acid (TNBS)(W. S. Hancock and J. E. Battersby, Anal. Biochem. (1976), 71, 260). Inthe case of a positive TNBS test (indicating incomplete coupling), thesuspension is allowed to react for another three times. This process isrepeated until the desired product has been achieved. After the removalof the last Fmoc protecting group, the resin is washed with DMF (3×),CH₂Cl₂ (3×) and DMF again (3×). The remaining free-amine group is thenacetylated using a cocktail of DIEA:Ac₂O (1:1) for 5 minutes at roomtemperature. Full-length peptides were then cleaved from solid supportusing TFA:TIS:H₂O (95:2.5:2.5) for 150 minutes, precipitated in coldethyl ether and lyophilized. The polymer was reconstituted in a 1:1solution of A:B (A: H₂O, 0.1% TFA) (B: 90:10:0.1 acetonitrile/H₂O/TFA).

The compositions described herein may be prepared by successive amidebond-forming procedures in which amide bonds are formed between theβ-amino group of a first β-amino acid residue or a precursor thereof andthe α-carboxyl group of a second β-amino acid residue or α-amino acidresidue or a precursor thereof. The amide bond-forming step may berepeated as many times, and with specific α-amino acid residues and/orβ-amino acid residues and/or precursors thereof, as required to give thedesired α/β-polypeptide. Also analogs comprising two, three, or moreamino acid residues (α- or β-) may be joined together to yield largeranalogs comprising any combination of α-, or β-amino acids. Cycliccompounds may be prepared by forming peptide bonds between theN-terminal and C-terminal ends of a previously synthesized linearpolypeptide or through the disulfide crosslinking of sidechains ofnon-adjacent residues. β³-amino acids may be produced enantioselectivelyfrom corresponding β-amino acids. For instance, by Arndt-Eiserthomologation of N-protected α-amino acids. Homologation may be followedby coupling of the reactive diazoketone intermediate of the Wolffrearrangement with a β-amino acid residue.

In some embodiments, the invention relates to All unique patterns of α-or β-amino acids residues from about two to about seven residues inlength are explicitly within the scope of the invention. In someembodiments, the composition comprises an analog, wherein the analogwherein the analog comprises a repetitive pattern of sequential β-aminoacids from the amino-terminus to the carboxy-terminus, and wherein theanalog is an agonist or antagonist of the receptor to which itselectively binds or associates. For instance, in some embodiments, theanalog is a VIP analog or a functional fragment thereof that selectivitybinds to VPAC1, VPAC2, or PAC1 and wherein the VIP analog of functionalfragment thereof is an agonist or antagonist of at least one receptorchosen from: VPAC1, VPAC2, and PAC1. In some embodiments, the methods oftreatment or prevention include administration of VIP analogs, whereinthe VIP analog is an agonist or antagonist of at least one receptorchosen from: VPAC1, VPAC2, and PAC1. In some embodiments, thecomposition comprises an analog, wherein the analog wherein the analogdoes not comprise a repetitive pattern of sequential β-amino acids fromthe amino-terminus to the carboxy-terminus chosen from the following:ααααααβ, αααααβα, ααααβαα, αααβααα, ααβαααα, αβααααα, βαααααα, αααααββ,ααααββα, αααββαα, ααββααα, αββαααα, ββααααα, βαααααβ, βααααβα, βαααβαα,βααβααα, βαααα, αβααααβ, αβαααβα, αβααβαα, αβαβααα, ααβαααβ, ααβααβα,ααβαβαα, αααβααβ, αααβαβα, and ααααβαβ. In some embodiments, thecomposition comprises an analog, wherein the analog does not comprises arepetitive pattern of sequential β-amino acids from the amino-terminusto the carboxy-terminus chosen from the following: βααβαααβααβαααβααα,βααβαααβααβαααββαα, βααβαααβααβαααβββα, and βααβαααβααβαααββββ. In someembodiments, the composition comprises an analog, wherein the analogthat does not comprise a repetitive pattern of sequential β-amino acidsfrom the amino-terminus to the carboxy-terminus chosen from thefollowing: ββαβαααβααβαααβααβ; βαββαααβααβαααβααβ; βααββααβααβαααβααβ;βααβαβαβααβαααβααβ; βααβααββααβαααβααβ; βααβαααββαβαααβααβ;βααβαααβαββαααβααβ; βααβαααβααββααβααβ; βααβαααβααβαβαβααβ;βααβαααβααβααββααβ; βααβαααβααβαααββαβ; and βααβαααβααβαααβαββ.

In some embodiments, the composition comprises an analog, wherein theanalog does not comprises a repetitive pattern of sequential β-aminoacids from the amino-terminus to the carboxy-terminus chosen from thefollowing: ββααβααβαααβααβααα; βαβαβααβαααβααβααα; βααββααβαααβααβααα;βαααββαβαααβααβααα; βαααβαββαααβααβααα; βαααβααββααβααβααα;βαααβααβαβαβααβααα; βαααβααβααββααβααα; βαααβααβαααββαβααα;βαααβααβαααβαββααα; βαααβααβαααβααββαα; βαααβααβαααβααβαβα; andβαααβααβαααβααβααβ.

In some embodiments, the composition comprises an analog, wherein theanalog doe not comprise a repetitive pattern of sequential β-amino acidsfrom the amino-terminus to the carboxy-terminus chosen from thefollowing: βααβαααβααβαααβααα, βααβαααβααβαααββαα, βααβαααβααβαααβββα,and βααβαααβααβαααββββ, wherein any α-amino acid residue may be anon-natural amino acid. In some embodiments, the composition comprisesan analog, wherein the analog comprises a repetitive pattern ofsequential β-amino acids from the amino-terminus to the carboxy-terminuschosen from the following: βααβαααβααβαααβααα, βααβαααβααβαααββαα,βααβαααβααβαααβββα, and βααβαααβααβαααββββ, wherein at least one α-aminoacid residue may be a non-natural amino acid. In some embodiments, thecomposition comprises an analog, wherein the analog comprises arepetitive pattern of sequential β-amino acids from the amino-terminusto the carboxy-terminus chosen from the following: βααβαααβααβαααβααα,βααβαααβααβαααββαα, βααβαααβααβαααβββα, and βααβαααβααβαααββββ, whereinfrom about 1 to about 10 α-amino acid residues may be a non-naturalamino acid. In any of the above-mentioned patterns one or more of theβ-amino acid residues may be replaced or modified with cyclic β-aminoacid (cyclically-constrained beta amino acid), such as APC or ACPC.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog is either: (a) an agonist of VPAC2 receptor; or (b) interfereswith VPAC2 receptor signaling pathway and comprises the followingrepetitive pattern of β-amino acids from the amino-terminus to thecarboxy terminus: β₁α₁α₂β₂α₃α₄α₅β₃, wherein β₁, β₂ and β₃ are β-homoamino acids; wherein there is an optional β amino acid (β₀ or β₄) to oneof the β-homo amino acids (e.g. β₁₋₃); wherein the composition,pharmaceutical compositions, kit, or polypeptides of the inventionoptionally comprise amino acids with a pattern selected from: ααααααβ,αααααβα, ααααβαα, αααβααα, ααβαααα, αβααααα, βαααααα, αααααββ, ααααββα,αααββαα, ααββααα, αββαααα, ββααααα, βαααααβ, βααααβα, βαααβαα, βααβααα,βαβαααα, αβααααβ, αβαααβα, αβααβαα, αβαβααα, ααβαααβ, ααβααβα, ααβαβαα,αααβααβ, αααβαβα, and ααααβαβ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog does not comprises a repetitive pattern of sequential β-aminoacids from the amino-terminus to the carboxy-terminus chosen from thefollowing: βααβαααβααβαααβααα; βααβαααβααβαααββαα; βααβαααβααβαααβββα;and βααβαααβααβαααββββ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog that does not comprise a pattern of sequential α or β-amino acidsfrom the amino-terminus to the carboxy-terminus chosen from thefollowing: ββαβαααβααβαααβααβ; βαββαααβααβαααβααβ; βααββααβααβαααβααβ;βααβαβαβααβαααβααβ; βααβααββααβαααβααβ; βααβαααββαβαααβααβ;βααβαααβαββαααβααβ; βααβαααβααββααβααβ; βααβαααβααβαβαβααβ;βααβαααβααβααββααβ; βααβαααβααβαααββαβ; and βααβαααβααβαααβαββ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog does not comprise a repetitive pattern of sequential β-aminoacids from the amino-terminus to the carboxy-terminus chosen from thefollowing: ββααβααβαααβααβααα; βαβαβααβαααβααβααα; βααββααβαααβααβααα;βαααββαβαααβααβααα; βαααβαββαααβααβααα; βαααβααββααβααβααα;βαααβααβαβαβααβααα; βαααβααβααββααβααα; βαααβααβαααββαβααα;βαααβααβαααβαββααα; βαααβααβαααβααββαα; βαααβααβαααβααβαβα; andβαααβααβαααβααβααβ.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog doe not comprise a repetitive pattern of sequential β-amino acidsfrom the amino-terminus to the carboxy-terminus chosen from thefollowing: βααβαααβααβαααβααα; βααβαααβααβαααββαα; βααβαααβααβαααβββα;and βααβαααβααβαααββββ; wherein any α-amino acid residue may be anon-natural amino acid.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog comprises a repetitive pattern of sequential β-amino acids fromthe amino-terminus to the carboxy-terminus chosen from the following:βααβαααβααβαααβαα; βααβαααβααβαααββαα; βααβαααβααβαααβββα; andβααβαααβααβαααββββ; wherein at least one α-amino acid residue may be anon-natural amino acid.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprise a VIP analog, wherein theanalog comprises a repetitive pattern of sequential β-amino acids fromthe amino-terminus to the carboxy-terminus chosen from the following:βααβαααβααβαααβααα; βααβαααβααβαααββαα; βααβαααβααβαααβββα; andβααβαααβααβαααββββ; wherein from about 1 to about 10 α-amino acidresidues may be a non-natural amino acid.

In any of the above-mentioned patterns one or more of the β-amino acidresidues may be replaced or modified with cyclic 3-amino acid(cyclically-constrained beta amino acid), such as APC or ACPC;

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition, pharmaceutical compositions, kit,or polypeptides of the invention comprises a VIP analog, comprisingFTENYTKLRK (Seq ID No. 228) wherein at least two adjacent amino acidsare replaced by two sequential β-amino acids. In some embodiments, thecomposition, pharmaceutical compositions, kits, or polypeptides of theinvention comprise a VIP analog, wherein the at least two adjacentβ-amino acids are x and y.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus: β₁α₁α₂β₂α₃α₄α₅β₃α₆α₇β₄α₈α₉α₁₀β₅α₁₁α₁₂β₆,wherein β₁=a beta-3 threonine or ACPC; α₁=an alpha arginine; α₂=an alphaleucine, β₂=a beta-3 arginine or APC; α₃=an alpha lysine; α₄=an alphaglutamine; α₅=an alpha leucine; β₃=a beta-3 alanine or ACPC; α₆=an alphavaline; α₇=an alpha lysine; β₄=a beta-3 lysine or APC; α₈=an alphatyrosine; α₉=an alpha leucine; α₁₀=an alpha asparagine; β₅=a beta-3alanine or ACPC; α₁₁=an alpha isoleucine; α₁₂=an alpha leucine, β₆=abeta-3 asparagine; wherein the repetitive pattern is, optionally,preceded by: HSDAVFTDNY if the composition comprises; and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus: β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃,wherein β₁=any beta amino acid; α₁=any alpha amino acid; α₂=any alphaamino acid; α₃=any alpha amino acid; β₂=any beta amino acid; α₄=anyalpha amino acid; α₅=any alpha amino acid; 133=any beta amino acid;α6=any alpha amino acid; α7=any alpha amino acid; α8=any alpha aminoacid; β₄=any beta amino acid; α₉=any alpha amino acid; α₁₀=any alphaamino acid; β₅=any beta amino acid; α₁₁=any alpha amino acid; α₁₂=anyalpha amino acid; α₁₃=any alpha amino acid; wherein the repetitivepattern is, optionally, preceded by: HSDAVFTDNY if the compositioncomprises, and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃,wherein β₁=a beta-3 threonine; α₁=any alpha amino acid; α₂=any alphaamino acid; α₃=any alpha amino acid; β₂=a beta-3 lysine; α₄=any alphaamino acid; α₅=any alpha amino acid; β₃=a beta-3 alanine; α6=any alphaamino acid; α7=any alpha amino acid; α8=any alpha amino acid; β₄=abeta-3 tyrosine; α₉=any alpha amino acid; α₁₀=any alpha amino acid; β₅=abeta-3 alanine; α₁₁=any alpha amino acid; α₁₂=any alpha amino acid;α₁₃=any alpha amino acid; wherein the repetitive pattern is, optionally,preceded by: HSDAVFTDNY if the composition comprises, and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃;wherein β₁=any beta amino acid; α₁=an alpha arginine, α₂=an alphaleucine; α₃=an alpha arginine; β₂=any beta amino acid; α₄=an alphaglutamine; α₅=an alpha leucine; β₃=any beta amino acid; β₆=an alphavaline acid; α₇=an alpha lysine; α₈=an alpha lysine; β₄=any beta aminoacid; α₉=an alpha leucine; α₁₀=an alpha asparagine; β₅=any beta aminoacid; α₁₁=an alpha isoleucine; α₁₂=an alpha leucine; α₁₃=an alphaasparagine; wherein the repetitive pattern is, optionally, preceded by:HSDAVFTDNY if the composition comprises, and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminusβ₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃β₆; wherein β₁=a beta-3tyrosine; α₁=any alpha amino acid; α₂=any alpha amino acid; α₃=an alphaamino acid; β₂=a beta-3 arginine; α₄=an alpha amino acid; α₅=any alphaamino acid; β₃=a beta-3 leucine; α₆=any alpha amino acid; α₇=any alphaamino acid; α₉=any alpha amino acid; β₄=a beta-3 lysine; α₉=any alphaamino acid; α₁₀=any alpha amino acid; β₅=a beta-3 asparagine; α₁₁=anyalpha amino acid; α₁₂=any alpha amino acid; α₁₃=any alpha amino acid;and β₆=a beta-3 asparagine; wherein the repetitive pattern is,optionally, preceded by: HSDAVFTDN, and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminusβ₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃β₆; wherein β₁=a beta-3tyrosine; α₁=an alpha threonine; α₂=an alpha arginine; α₃=an alphaleucine; β₂=a beta-3 arginine or APC; α₄=an alpha lysine; α₅=an alphaglutamine; β₃=a beta-3 leucine or ACPC; α₆=an alpha alanine; α₇=an alphavaline; α₈=an alpha lysine; β₄=a beta-3 lysine or APC; α₉=an alphatyrosine; α₁₀=an alpha leucine; β₅=a beta-3 asparagine or ACPC; α₁₁=analpha alanine; α₁₂=an alpha isoleucine; α₁₃=an alpha leucine; and β₆=abeta-3 asparagine; wherein the repetitive pattern is, optionally,preceded by: HSDAVFTDN, and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminusβ₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃β₆; wherein β₁=a beta-3tyrosine; α₁=an alpha threonine; α₂=an alpha arginine; α₃=an alphaleucine; β₂=a beta-3 arginine or APC; α₄=an alpha lysine; α₅=an alphaglutamine; β₃=a beta-3 leucine or ACPC; α₆=an alpha alanine; α₇=an alphavaline; α₈=an alpha lysine; β₄=a beta-3 lysine or APC; α₉=an alphatyrosine; α₁₀=an alpha leucine; β₅=a beta-3 asparagine or ACPC; α₁₁=analpha alanine; α₁₂=an alpha isoleucine; α₁₃=an alpha leucine; and β₆=abeta-3 asparagine; wherein the repetitive pattern is, optionally,preceded by: HSDAVFTDN;

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.

In some embodiments, the composition comprises HSDAVFTDN or HSDAVFTDNY,wherein at least one of the amino acids from HSDAVFTDN or HSDAVFTDNY arenon-natural or beta amino acids. In some embodiments, the compositioncomprises HSDAVFTDN or HSDAVFTDNY, wherein at least one of the aminoacids from HSDAVFTDN or HSDAVFTDNY is a beta-3, beta-2, cyclic, orheterocyclic beta amino acids. In some embodiments, the C-terminus isnot amidated. In some embodiments, the N-terminus is not acylated. Insome embodiments, the composition comprises HSDAVFTDN or HSDAVFTDNY,wherein the amino acids from HSDAVFTDN or HSDAVFTDNY are alpha aminoacids. In some embodiments, the composition comprises HSDAVFTDN orHSDAVFTDNY, wherein the amino acids from HSDAVFTDN or HSDAVFTDNY are notalpha amino acids. In some embodiments, the composition comprisesHSDAVFTDN or HSDAVFTDNY, wherein none of the amino acids from HSDAVFTDNor HSDAVFTDNY are beta-3 amino acids. In some embodiments, thecomposition comprises HSDAVFTDN or HSDAVFTDNY, wherein none of the aminoacids from HSDAVFTDN or HSDAVFTDNY are beta-2 amino acids. In someembodiments, the composition comprises HSDAVFTDN or HSDAVFTDNY, whereinnone of the amino acids from HSDAVFTDN or HSDAVFTDNY are ACPC or APC. Insome embodiments, the composition comprises HSDAVFTDN or HSDAVFTDNY,wherein none of the amino acids from HSDAVFTDN or HSDAVFTDNY are cyclic.In some embodiments, the composition comprises HSDAVFTDN or HSDAVFTDNY,wherein none of the amino acids from HSDAVFTDN or HSDAVFTDNY areheterocyclic.

“Selective” or “Selectivity” means that the analog of the presentinvention has a binding preference for one protein as compared toanother protein. In some embodiments, the binding preference may bemeasured as an affinity for a protein in terms of half maximalinhibitory concentration (IC50). In some embodiments, the bindingpreference may be measured as an affinity for a protein in terms of halfmaximal effective concentration (EC50). For example, an analog selectiveto VPAC2 receptor with a selectivity to VPAC2 means that the analog maybind to VPAC1 receptor but has a higher binding affinity for a domain ofthe VPAC2 receptor if the analog is exposed to both VPAC1 and VPAC2 atsimilar or equivalent concentrations. As used herein, an analog thatselectively binds to VPAC2 refers to an analog with increasedselectivity for the VPAC2 receptor compared to other known receptors orproteins to which the peptide may bind. In some embodiments, the analogselective for VPAC2 may be an agonist of the VPAC2 receptor peptide. Insome embodiments, the analog selective for VPAC2 may be an antagonist ofVPAC2 receptor. In some embodiments, an analog selective to VPAC2receptor means that the analog may bind to VPAC1 receptor but has ahigher binding affinity for a domain of the VPAC2 receptor if the analogis exposed to PAC1, VPAC1 receptor and VPAC2 receptors at similar orequivalent concentrations. In some embodiments, an analog selective toVPAC1 receptor means that the analog may bind to a domain of VPAC2 orPAC1 receptor but has a higher binding affinity for a domain of theVPAC1 receptor if the analog is exposed to PAC1, VPAC1 receptor andVPAC2 receptors at similar or equivalent concentrations. As used herein,an analog that selectively binds to VPAC1 refers to an analog withincreased selectivity for the VPAC1 receptor compared to other knownreceptors or proteins to which the peptide may bind. In someembodiments, the analog selective for VPAC1 may be an agonist of theVPAC1 receptor peptide. In some embodiments, the analog selective forVPAC1 may be an antagonist of VPAC1 receptor. In some embodiments, ananalog selective to VPAC1 receptor means that the analog may bind toVPAC2 receptor but has a higher binding affinity for a domain of theVPAC1 receptor if the analog is exposed to both VPAC1 receptor and VPAC2receptor at similar or equivalent concentrations. As used herein, ananalog that selectively binds to PAC1 refers to an analog with increasedselectivity for the PAC1 receptor as compared to other known receptorsor proteins to which the peptide may bind. In some embodiments, theanalog selective for PAC1 may be an agonist of the PAC1 receptorpeptide. In some embodiments, the analog selective for PAC1 may be anantagonist of PAC1 receptor. In some embodiments, an analog selective toPAC1 receptor means that the analog may bind to VPAC2 or VPAC1 receptorsbut has a higher binding affinity for a domain of the PAC1 receptor ifthe analog is exposed to PAC1, VPAC1 receptor and VPAC2 receptors atsimilar or equivalent concentrations. The degree of selectivity may bedetermined by a ratio of VPAC2 receptor binding affinity to VPAC1receptor binding affinity or by a ratio of VPAC2 receptor bindingaffinity to PAC1 receptor binding affinity. Binding affinity isdetermined as described below in Example 1.

In any of the embodiments described below wherein the polypeptidecomprises a residue designated f, the residue designated f is D-Phe orL-Phe or S. In some embodiments, the invention relates to compositionsor pharmaceutical compositions comprising a VIP analog, wherein theanalog comprises an α-amino acid and at least one β-amino acid, andwherein the analog is between 75% and 99% homologous toHfDAVFTNSYRKVLKRLSARKLLQDIL; where residue designated f (position 2) isD-Phe, and wherein the analog interferes with the VPAC1 receptorsignaling pathway. In some embodiments, the invention relates tocompositions or pharmaceutical compositions comprising a VIP analog,wherein the analog comprises an α-amino acid and at least one β-aminoacid, and wherein the analog is between 75% and 99% homologous toHfDAVFTNSYRKVLKRLSARKLLQDIL, where residue designated f (position 2) isD-Phe, and wherein the analog is an antagonist of the VPAC1 receptor. Insome embodiments, the composition comprises a VIP analog is from about80% to about 99% homologous to HfDAVFTNSYRKVLKRLSARKLLQDIL, whereresidue designated f (position 2) is D-Phe. In some embodiments the VIPanalog is from about 80% to about 85% homologous toHfDAVFTNSYRKVLKRLSARKLLQDIL, where residue designated f (position 2) isD-Phe. In some embodiments the VIP analog is from about 85% to about 90%homologous to HfDAVFTNSYRKVLKRLSARKLLQDIL, where residue designated f(position 2) is D-Phe. In some embodiments the VIP analog is from about90% to about 95% homologous to HfDAVFTNSYRKVLKRLSARKLLQDIL, whereresidue designated f (position 2) is D-Phe. In some embodiments the VIPanalog is from about 95% to about 99% homologous toHfDAVFTNSYRKVLKRLSARKLLQDIL, where residue designated f (position 2) isD-Phe. In some embodiments the VIP analog is about 95%, 96%, 97%, 98%,or 99% homologous to HfDAVFTNSYRKVLKRLSARKLLQDIL, where residuedesignated f (position 2) is D-Phe. In some embodiments, the compositionor pharmaceutical compositions comprise a VIP analog, wherein the analogis either: (a) an antagonist of VPAC1 receptor; or (b) interferes withVPAC1 receptor signaling pathway and comprises the following repetitivepattern of sequential β-amino acids from the amino-terminusβ₁α₁α₂β₂α₃α₄α₅β₃α₆α₇β₄α₈α₉α₁₀β₅, wherein β₁=any beta amino acid; α1=anyalpha amino acid; α2=any alpha amino acid; β₂=any beta amino acid;α₃=any alpha amino acid; α₄=any alpha amino acid; α₅=any alpha aminoacid; β₃=any beta amino acid; α₆=any alpha amino acid; α₇=any alphaamino acid; β₄=any beta amino acid, α₈=any alpha amino acid; α₉=anyalpha amino acid; α₁₀=any alpha amino acid; β₅=any beta amino acid;wherein the repetitive pattern is, optionally, preceded by: HfDAV FTNSY;and

-   -   wherein residue designated f (position 2) is D-Phe    -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.        In some embodiments, the composition or pharmaceutical        compositions comprise a VIP analog, wherein the analog is        either: (a) an antagonist of VPAC1 receptor; or (b) interferes        with VPAC1 receptor signaling pathway and comprises the        following repetitive pattern of sequential β-amino acids from        the amino-terminus β₁α₁α₂β₂α₃α₄α₅β₃α₆α₇β₄α₈α₉α₁₀β₅, wherein        β₁=any beta 3 amino acid; α₁=any alpha amino acid; α₂=any alpha        amino acid; β₂=any beta 3 amino acid; α₃=any alpha amino acid;        α₄=any alpha amino acid; α₅=any alpha amino acid; β₃=any beta 3        amino acid; α₆=any alpha amino acid; α₇=any alpha amino acid;        β₄=any beta 3 amino acid, α₈=any alpha amino acid; α₉=any alpha        amino acid; α₁₀=any alpha amino acid; β₅=any beta 3 amino acid;        wherein the repetitive pattern is, optionally, preceded by:        HfDAV FTNSY, and    -   wherein residue designated f (position 2) is D-Phe    -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.        In some embodiments, the composition or pharmaceutical        compositions comprise a VIP analog, wherein the analog is        either: (a) an antagonist of VPAC1 receptor; or (b) interferes        with VPAC1 receptor signaling pathway and comprises the        following repetitive pattern of sequential β-amino acids from        the amino-terminus β₁α₁α₂β₂α₃α₄α₅β₃α₆α₇β₄α₈α₉α₁₀β₅, wherein β₁=a        beta-3 arginine; α₁=any alpha amino acid; α₂=any alpha amino        acid; β₂=a beta-3 leucine; α₃=any alpha amino acid; α₄=any alpha        amino acid; α₅=any alpha amino acid; β₃=a beta-3 serine; α₆=any        alpha amino acid; α₇=any alpha amino acid; β₄=a beta-3 lysine,        α₈=any alpha amino acid; α₉=any alpha amino acid; α₁₀=any alpha        amino acid; β₅=a beta-3 aspartic acid; wherein the repetitive        pattern is, optionally, preceded by: HfDAV FTNSY; and    -   wherein residue designated f (position 2) is D-Phe    -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof.        In some embodiments, the composition comprises HfDAVFTDN or        HfDAVFTDNY, wherein at least one of the amino acids from        HfDAVFTDN or HfDAVFTDNY are non-natural or beta amino acids,        wherein residue designated f (position 2) is D-Phe. In some        embodiments, the composition comprises HfDAVFTDN or HfDAVFTDNY,        wherein at least one of the amino acids from HfDAVFTDN or        HfDAVFTDNY is a beta-3, beta-2, cyclic, or heterocyclic beta        amino acids, and wherein residue designated f (position 2) is        D-Phe. In some embodiments, the C-terminus is not amidated. In        some embodiments, the N-terminus is not acylated. In some        embodiments, the composition comprises HfDAVFTDN or HfDAVFTDNY,        wherein the amino acids from HfDAVFTDN or HfDAVFTDNY are alpha        amino acids, and wherein residue designated f (position 2) is        D-Phe. In some embodiments, the composition comprises HfDAVFTDN        or HfDAVFTDNY, wherein the amino acids from HfDAVFTDN or        HfDAVFTDNY are not alpha amino acids, and wherein residue        designated f (position 2) is D-Phe. In some embodiments, the        composition comprises HfDAVFTDN or HfDAVFTDNY, wherein none of        the amino acids from HfDAVFTDN or HfDAVFTDNY are beta-3 amino        acids, and wherein residue designated f (position 2) is D-Phe.        In some embodiments, the composition comprises HfDAVFTDN or        HfDAVFTDNY, wherein none of the amino acids from HfDAVFTDN or        HfDAVFTDNY are beta-2 amino acids, and wherein residue        designated f (position 2) is D-Phe. In some embodiments, the        composition comprises HfDAVFTDN or HfDAVFTDNY, wherein none of        the amino acids from HfDAVFTDN or HfDAVFTDNY are ACPC or APC,        and wherein residue designated f (position 2) is D-Phe. In some        embodiments, the composition comprises HfDAVFTDN or HfDAVFTDNY,        wherein none of the amino acids from HfDAVFTDN or HfDAVFTDNY are        cyclic, wherein residue designated f (position 2) is D-Phe. In        some embodiments, the composition comprises HfDAVFTDN or        HfDAVFTDNY, wherein none of the amino acids from HfDAVFTDN or        HfDAVFTDNY are heterocyclic, and wherein residue designated f        (position 2) is D-Phe.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus: β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅; whereinβ₁=any beta amino acid; α₁=any alpha amino acid; α₂=any alpha aminoacid; α₃=an alpha amino acid, β₂=any beta amino acid; α₄=an alpha aminoacid; α₅=any alpha amino acid; β₃=any beta amino acid; α₆=any alphaamino acid; α₇=any alpha amino acid; α₈=any alpha amino acid; β₄=anybeta amino acid; α₉=any alpha amino acid; α₁₀=any alpha amino acid;β₅=any beta amino acid; wherein the repetitive pattern is, optionally,preceded by: HfDAV FTNSY or HfDAV FTNS; and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof; and wherein residue designated        f (position 2) is D-Phe.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅; whereinβ₁=a beta-3 arginine or beta-3 tyrosine; α₁=any alpha amino acid; α₂=anyalpha amino acid; α₃=an alpha amino acid; β₂=a beta-3 lysine or beta-3leucine; α₄=an alpha amino acid; α₅=any alpha amino acid; β₃=a beta-3serine or a beta-3 leucine; α₆=any alpha amino acid; α₇=any alpha aminoacid; α₈=any alpha amino acid; β₄=a beta-3 leucine or beta-3 lysine;α₉=any alpha amino acid; α₁₀=any alpha amino acid; β₅=a beta-3 asparticacid or beta-3 glutamine; wherein the repetitive pattern is, optionally,preceded by: HfDAV FTNSY or HfDAV FTNS; and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof, and wherein residue designated        f (position 2) is D-Phe.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅; whereinβ₁=a beta-3 arginine; beta-3 tyrosine, or APC; α₁=any alpha amino acid;α₂=any alpha amino acid; α₃=an alpha amino acid; β₂=ACPC or APC; α₄=analpha amino acid; α₅=any alpha amino acid; β₃=ACPC or a beta-3 leucine;α₆=any alpha amino acid; α₇=any alpha amino acid; α₈=any alpha aminoacid; β₄=a beta-3 leucine, beta-3 lysine, or APC; α₉=any alpha aminoacid; α₁₀=any alpha amino acid; β₅=a beta-3 aspartic acid or ACPC;wherein the repetitive pattern is, optionally, preceded by: HfDAV FTNSYor HfDAV FTNS; and

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is, optionally, acylated;    -   or functional fragments thereof, and wherein residue designated        f (position 2) is D-Phe.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HfDAV FTNSY Z KV X K RL X AR K LLQ D  IL HfDAV FTNSY R KV X K RL X AR ZLLQ D  IL HfDAV FTNSY R KV X K RL X AR K LLQ X  IL HfDAV FTNSY Z KV X KRL X AR Z LLQ X  IL HfDAV FTNSY R KVL Z  RL X AR K L LQ X  IL HfDAVFTNSY Z KVL Z  RL X AR K L LQ X  IL HfDAV FTNS Y  RKV X K R L SAR Z LL XD IL HfDAV FTNS Y  RKV X K R X SAR K LL X D IL HfDAV FTNS Y  RKV X K R XSAR Z LL X D ILwherein residue designated f (position 2) is D-Phe, wherein eachunderlined residue is a beta amino acid, wherein X is a ACPC, wherein Zis APC, and wherein the analog interferes with the VPAC1 receptorsignaling pathway. In some embodiments, the invention relates tocompositions or pharmaceutical compositions comprising a VIP analog,wherein the analog comprises an α-amino acid and at least one β-aminoacid, and wherein the analog is between 75% and 100% homologous to oneor more of the following sequences:

HfDAV FTNSY Z KV X K RL X AR K LLQ D  IL HfDAV FTNSY R KV X K RL X AR ZLLQ D  IL HfDAV FTNSY R KV X K RL X AR K LLQ X  IL HfDAV FTNSY Z KV X KRL X AR Z LLQ X  IL HfDAV FTNSY R KVL Z  RL X AR K L LQ X  IL HfDAVFTNSY Z KVL Z  RL X AR K L LQ X  IL HfDAV FTNS Y  RKV X K R L SAR Z LL XD IL HfDAV FTNS Y  RKV X K R X SAR K LL X D IL HfDAV FTNS Y  RKV X K R XSAR Z LL X D ILwherein residue designated f (position 2) is D-Phe, wherein eachunderlined residue is a beta amino acid, wherein X is a ACPC, wherein Zis APC, and wherein the analog is an antagonist of the VPAC1 receptor;or functional fragments thereof.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAV FTDNY TRLRK QlAVK KYLN a  ILN HSDAV FTDNY t RL r K QL a VK k YLN a I ln HSDAV FTDNY t RLR k  QL a VK K y LN a  ILN HSDAV FTDN y  TRL r K Ql AVK k YL n A I ln HSDAV FTDNY t RL z K QL x VK k YLN x  IL n HSDAVFTDNY t RL z K QL x VK z YLN x  I ln HSDAV FTDNY x RL z K QL x VK k YLNx  I ln HSDAV FTDNY x RL z K QL x VK z YLN x  I ln HSDAV FTDNY t RLR z QL x VK K y LN x  ILN HSDAV FTDNY x RLR z  QL x VK K y LN x  ILN HSDAVFTDN y  TRL z K Q l AVK z YL x A I ln HSDAV FTDN y  TRL z K Q x AVK k YLx A I ln HSDAV FTDN y  TRL z K Q x AVK z YL x A I lnwherein each underlined residue is a beta amino acid corresponding tothe single code amino acid upon which it is based, wherein X is a ACPC,and wherein Z is APC; or functional fragments thereof.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 99% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY, and wherein the analog stimulates theVPAC2 receptor signaling pathway. In some embodiments, the inventionrelates to compositions or pharmaceutical compositions comprising a VIPanalog, wherein the analog comprises an α-amino acid and at least oneβ-amino acid, and wherein the analog is between 75% and 99% homologousto HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY, wherein the analog is an agonist ofthe VPAC2 receptor. In some embodiments, the composition comprises a VIPanalog is from about 80% to about 99% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY. In some embodiments the VIP analog isfrom about 80% to about 85% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY. In some embodiments the VIP analog isfrom about 85% to about 90% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY. In some embodiments the VIP analog isfrom about 90% to about 95% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY. In some embodiments the VIP analog isfrom about 95% to about 99% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY. In some embodiments the VIP analog isabout 95%, 96%, 97%, 98%, or 99% homologous toHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY. In some embodiments the VIP analog isHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY.

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is unmodified or modified; or functional        fragments thereof.

In some embodiments, the composition comprises a VIP analog, wherein theanalog comprises the following repetitive pattern of sequential β-aminoacids from the amino-terminus:β₁α₁α₂α₃β₂α₄α₅β₃α₆α₇α₈β₄α₉α₁₀β₅α₁₁α₁₂α₁₃β₆; wherein β₁=a beta-3threonine or a beta-3 tyrosine; α₁=any alpha amino acid; α₂=any alphaamino acid; α₃=an alpha amino acid, β₂=a beta-3 lysine or a beta-3arginine; α₄=an alpha amino acid; α₅=any alpha amino acid; β₃=a beta-3alanine or a beta-3 valine; α₆=any alpha amino acid; α₇=any alpha aminoacid; α₈=any alpha amino acid; β₄=a beta-3 tyrosine or a beta-3 lysine;α₉=any alpha amino acid; α₁₀=any alpha amino acid; β₅=a beta-3 serine ora beta-3 glutamine; α₁₁=any alpha amino acid; α₁₂=any alpha amino acid;α₁₃=any alpha amino acid; and β₆=a beta-3 lysine or a beta-3 asparagine;and wherein the repetitive pattern is, optionally, preceded by: HSDAVFTDNY or HSDAV FTDN, and wherein the repetitive pattern is, optionally,succeeded by: K, KR, or KRY.

-   -   wherein the C-terminus is, optionally, amidated; and    -   wherein the N-terminus is unmodified or modified; or functional        fragments thereof;    -   and wherein the analog or functional fragment thereof is a VPAC2        agonist.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAVFTDNY X RL Z KQV X AK K YLQ S IK N KRY HSDAVFTDNY T RL Z KQV X AK ZYLQ S IK N KRY HSDAVFTDNY T RL Z KQV X AK K YLQ X IK N KRY HSDAVFTDNY XRL Z KQV X AK Z YLQ X IK X KRY HSDAVFTDNY T RLR Z QV X AKK Y LQ X IKN KRY HSDAVFTDNY X RLR Z QV X AKK Y LQ X IKN K RY HSDAVFTDN Y TRL Z KQ VSAK Z YL X SIK N KRY HSDAVFTDN Y TRL Z KQ X SAK K YL X SIK N KRYHSDAVFTDN Y TRL Z KQ X SAK Z YL X SIK N KRYwherein each underlined residue is a beta amino acid corresponding tothe single code amino acid upon which it is based, wherein X is a ACPC,and wherein Z is APC; or functional fragments thereof; wherein theC-terminus is, optionally, amidated; and wherein the N-terminus isunmodified.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAVFTDNY X RL Z KQV X AK K YLQ S IK N KRY HSDAVFTDNY T RL Z KQV X AK ZYLQ S IK N KRY HSDAVFTDNY T RL Z KQV X AK K YLQ X IK N KRY HSDAVFTDNY XRL Z KQV X AK Z YLQ X IK X KRY HSDAVFTDNY T RLR Z QV X AKK Y LQ X IKN KRY HSDAVFTDNY X RLR Z QV X AKK Y LQ X IKN K RY HSDAVFTDN Y TRL Z KQ VSAK Z YL X SIK N KRY HSDAVFTDN Y TRL Z KQ X SAK K YL X SIK N KRYHSDAVFTDN Y TRL Z KQ X SAK Z YL X SIK N KRYwherein each underlined residue is a beta amino acid corresponding tothe single code amino acid upon which it is based, wherein X is a ACPC,and wherein Z is APC; or functional fragments thereof; wherein theC-terminus is, optionally, amidated; and wherein the N-terminus is,optionally, modified.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAVFTDNY X RL Z KQV X AK K YLQ S IK N KRY HSDAVFTDNY T RL Z KQV X AK ZYLQ S IK N KRY HSDAVFTDNY T RL Z KQV X AK K YLQ X IK N KRY HSDAVFTDNY XRL Z KQV X AK Z YLQ X IK X KRY HSDAVFTDNY T RLR Z QV X AKK Y LQ X IKN KRY HSDAVFTDNY X RLR Z QV X AKK Y LQ X IKN K RY HSDAVFTDN Y TRL Z KQ VSAK Z YL X SIK N KRY HSDAVFTDN Y TRL Z KQ X SAK K YL X SIK N KRYHSDAVFTDN Y TRL Z KQ X SAK Z YL X SIK N KRYwherein each underlined residue is a beta amino acid corresponding tothe single code amino acid upon which it is based, wherein X is a ACPC,and wherein Z is APC; or functional fragments thereof; wherein theC-terminus is, optionally, amidated; wherein the N-terminus is,optionally, modified; and wherein the VIP analog or functional fragmentthereof is a VPAC2 agonist.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAV FTDNY T RL R K QV A AK K YLQ S  IK N KR Y HSDAV FTDNY T RLR K  QVA AK K Y LQ S  IKN K R Y HSDAV FTDN Y  TRL R K Q V AAK K YL Q S IK N KRY HSDAV FTDNY X RL Z K QV X AK K YLQ S  IK N KR Y HSDAV FTDNY T RL Z KQV X AK Z YLQ S  IK N KR Y HSDAV FTDNY T RL Z K QV X AK K YLQ X  IK N KRY HSDAV FTDNY X RL Z K QV X AK Z YLQ X  IK X KR Y HSDAV FTDNY T RLR Z QV X AK K Y LQ X  IKN K R Y HSDAV FTDNY X RLR Z  QV X AK K Y LQ X  IKNK R Y HSDAV FTDN Y  TRL Z K Q V AAK Z YL X S IK N KR Y HSDAV FTDN Y  TRLZ K Q X AAK K YL X S IK N KR Y HSDAV FTDN Y  TRL Z K Q X AAK Z YL X S IKN KR Ywherein each underlined residue is an unnatural amino acid correspondingto the single code amino acid upon which it is based, wherein X is aACPC, and wherein Z is APC; or functional fragments thereof; wherein theC-terminus is, optionally, amidated; wherein the N-terminus is,optionally, modified; and wherein the VIP analog or functional fragmentthereof is a VPAC1 agonist.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAV FTDNY T RL R K QV A AK K YLQ S  IK N KR Y HSDAV FTDNY T RLR K  QVA AK K Y LQ S  IKN K R Y HSDAV FTDN Y  TRL R K Q V AAK K YL Q S IK N KRY HSDAV FTDNY X RL Z K QV X AK K YLQ S  IK N KR Y HSDAV FTDNY T RL Z KQV X AK Z YLQ S  IK N KR Y HSDAV FTDNY T RL Z K QV X AK K YLQ X  IK N KRY HSDAV FTDNY X RL Z K QV X AK Z YLQ X  IK X KR Y HSDAV FTDNY T RLR Z QV X AK K Y LQ X  IKN K R Y HSDAV FTDNY X RLR Z  QV X AK K Y LQ X  IKNK R Y HSDAV FTDN Y  TRL Z K Q V AAK Z YL X S IK N KR Y HSDAV FTDN Y  TRLZ K Q X AAK K YL X S IK N KR Y HSDAV FTDN Y  TRL Z K Q X AAK Z YL X S IKN KR Ywherein each underlined residue is a beta amino acid corresponding tothe single code amino acid upon which it is based, wherein X is a ACPC,and wherein Z is APC; or functional fragments thereof; wherein theC-terminus is, optionally, amidated; wherein the N-terminus is,optionally, modified; and wherein the VIP analog or functional fragmentthereof is a VPAC1 agonist.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog is between 75% and 100% homologous to one or more of thefollowing sequences:

HSDAV FTDNY T RL R K QV A AK K YLQ S  IK N KR Y HSDAV FTDNY T RLR K  QVA AK K Y LQ S  IKN K R Y HSDAV FTDN Y  TRL R K Q V AAK K YL Q S IK N KRY HSDAV FTDNY X RL Z K QV X AK K YLQ S  IK N KR Y HSDAV FTDNY T RL Z KQV X AK Z YLQ S  IK N KR Y HSDAV FTDNY T RL Z K QV X AK K YLQ X  IK N KRY HSDAV FTDNY X RL Z K QV X AK Z YLQ X  IK X KR Y HSDAV FTDNY T RLR Z QV X AK K Y LQ X  IKN K R Y HSDAV FTDNY X RLR Z  QV X AK K Y LQ X  IKNK R Y HSDAV FTDN Y  TRL Z K Q V AAK Z YL X S IK N KR Y HSDAV FTDN Y  TRLZ K Q X AAK K YL X S IK N KR Y HSDAV FTDN Y  TRL Z K Q X AAK Z YL X S IKN KR Ywherein each underlined residue is a beta-3 homo amino acidcorresponding to the single code amino acid upon which it is based,wherein X is a ACPC, and wherein Z is APC; or functional fragmentsthereof; wherein the C-terminus is, optionally, amidated; wherein theN-terminus is, optionally, modified; and wherein the VIP analog orfunctional fragment thereof is a VPAC1 agonist.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog, wherein the analogcomprises an α-amino acid and at least one β-amino acid, and wherein theanalog comprises an amino acid sequence that is between 75% and 100%homologous to one or more of the following sequences:

1155 HSDAVFTENYTKLRKQLA x KKY x ND l KKG g T 1156 HSDAVFTENYTKLRKQLAA zKY x NDL k KG g T 1157 HSDAVFTENYTKLRKQ x AA z KYL x DL k KGGT 1185HBDAVFTENYTKLRKQLAA z KY x NDL k KG g T 1186 HBDA v FTENYTKLRKQLAA z KYx NDL k KG g T 1187 HBDA v FTE n YTKLRKQLAA z KY x NDL k KG g T 1188 H xDAVFTENYTKLRKQLAA z KY x NDL k KG g T 1189 H x DA x FTENYTKLRKQLAA z KYx NDL k KG g T 1190 H x DA x FTE x YTKLRKQLAA z KY x NDL k KG g T 1211 Hx DAVFTDNY t RLR k QLA v KKY l NAI l N 1212 H x DA x FTDNY t RLR k QLA vKKY l NAI l N 1213 HF F DAVFTDNY t RLR k QLA v KKY l NAI l N 1214 HF FDA x FTDNY t RLR k QLA v KKY l NAI l N 1215 H x DA v FTDNY t RLR k QLA vKKY l NAI l N 1216 HF F DA v FTDNY t RLR k QLA v KKY l NAI l N 1217 H sDA v FTDNY t RLR k QLA v KKY l NAI l N 1218 H s DA v FTDnY t RLR k QLA vKKY l NAI l N 1219 H F FDA v FTD n Y t RLR k QLA v KKY l NAI l N 1220 HFd AVF t DNY t RLR k QLA v KKY l NAI l N 1221 HS d AVF t DNY t RLR k QLAv KKY l NAI l N 1201 H x DA v FTENYTKLRKQLAA z KY x NDL k KG g T 1202 Hx DA v FTENYTKLRKQ l AA z KY x NDL k KG g T 1203 H x DA v FTENYTKLRK gLAA z KY x NDL k KG g T 1204 H x DA v FTENYTKLR k QLAA z KY x NDL k KG gT 1205 H x DA v FTENYTKL r KQLAA z KY x NDL k KG g T 1206 H x DA vFTENYTK l RKQLAA z KY x NDL k KG g T 1207 H x DA x FTE ny TKLRKQLAA z KYx NDL k KG g T 1208 H x DA x FTE xy TKLRKQ l AA z KY x NDL k KG g T 1209H x DA x FTE xy TKLRK g LAA z KY x NDL k KG g T 1210 H x DA x FTE xyTKLR k QLAA z KY x NDL k KG g Twherein each underlined residue is any unnatural amino acid; or anybeta-2 amino acid; any beta-3 amino acid; or a beta-3 homo amino acidcorresponding to the single code amino acid upon which it is based;wherein X is a ACPC, and wherein Z is APC; or functional fragmentsthereof; wherein the C-terminus is, optionally, amidated; wherein theN-terminus is, optionally, modified; and wherein the VIP analog orfunctional fragment thereof is a VPAC₂R agonist. In some embodiments,the VIP analog is a VPAC1 agonist that selectivity binds VPAC₁R at leastone order of magnitude greater than its selectivity for VPAC₂R. In someembodiments, the VIP analog is a VPAC₂R agonist that selectivity bindsVPAC₂R at least one order of magnitude greater than its selectivity forVPAC₁R. In some embodiments the EC50 of the disclosed analogs hereinresults in greater selectivity for the specifically targeted receptorthan for any of the disclosed receptors herein. In some embodiments,this selectivity is significantly greater than that of the naturalreceptor ligand.

In some embodiments, the invention relates to compositions orpharmaceutical compositions comprising a VIP analog or pharmaceuticalsalts thereof, wherein the analog comprises an α-amino acid and at leastone β-amino acid, and wherein the analog comprises an amino acidsequence that is between 75% and 100% homologous to any of the aminoacid sequences provided in this application.

The invention relates to methods of manufacturing a compositioncomprising an analog, wherein the analog comprises any of thecompositions disclosed herein. In some embodiments, the inventionrelates to methods of manufacturing a composition comprising an analog,wherein the analog comprises an α-amino acid, at least one β-amino acid,and at least one modified amino acid residue comprising ACPC or APC. Theinvention relates to methods of manufacturing a composition comprising aVIP family analog, wherein the VIP family analog comprises an α-aminoacid and at least one β-amino acid. The invention relates to methods ofmanufacturing a composition comprising a VIP analog, wherein the VIPanalog comprises an α-amino acid and at least one β-amino acid. Themethod used to fabricate polypeptide compounds may be any means ofpolypeptide synthesis. Using methods of peptide synthesis, polypeptidesfabricated according to the present method are generally less than about100 residues long. In some embodiments, the invention relates to amethod of manufacturing an analog (or fragments herein) comprisingnon-natural amino acids from from about 5 total residues to about 50total residues, from about 10 total residues to about 20 total residues,from about 20 total residues to about 30 total residues, from about 30total residues to about 40 total residues, from about 40 total residuesto about 50 total residues, from about 50 to about 60 total residues,from about 60 to about 70 total residues from about 70 to about 80 totalresidues, from about 80 to about 90 total residues, and from about 90 toabout 100 total residues. Ranges above and below these stated ranges arewithin the scope of the invention. Many commercial services, such asAbgent (San Diego, Calif., USA) offer peptide synthesis services up toabout 100 residues. In some embodiments, the invention relates to amethod of manufacturing an analog comprising no more than 100non-natural amino acids. In some embodiments, the invention relates to amethod of manufacturing an analog comprising no more than 90 non-naturalamino acids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 80 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 70 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 60 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 50 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 40 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 30 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 20 non-natural aminoacids. In some embodiments, the invention relates to a method ofmanufacturing an analog comprising no more than 10 non-natural aminoacids. In some embodiments, the method of manufacturing the analogcomprises synthesizing the analog using at least one, and, in someembodiments, a plurality of the following non-naturally occurring aminoacid residues: (2S,3R)-3-(amino)-2-hydroxy-4-(4-nitrophenyl)butyricacid, (2R,3R)-3-(amino)-2-hydroxy-4-phenylbutyric acid,(R)-3-(amino)-5-phenylpentanoic acid,(R)-3-(amino)-4-(2-naphthyl)butyric acid, (R)-2-methyl-β-Phe-OH,(R)-3,4-dimethoxy-β-Phe-OH, (R)-(3-pyridyl)-β-Ala-OH,(R)-3-(trifluoromethyl)-β-Phe-OH, (R)-3-cyano-β-Phe-OH,(R)-3-methoxy-β-Phe-OH, (R)-3-methyl-β-Phe-OH,(R)-4-(4-pyridyl)-β-HomoAla-OH, (R)-4-(trifluoromethyl)-β-HomoPhe-OH,(R)-4-(trifluoromethyl)-β-Phe-OH, (R)-4-bromo-β-Phe-OH,(R)-4-chloro-β-HomoPhe-OH, (R)-4-chloro-β-Phe-OH,(R)-4-cyano-β-HomoPhe-OH, (R)-4-cyano-β-Phe-OH, (R)-4-fluoro-β-Phe-OH,(R)-4-methoxy-β-Phe-OH, (R)-4-methyl-β-Phe-OH, (R)-β-Tyr-OH,(R)-4-β-pyridyl)-β-HomoAla-OH, (R)-4-fluoro-β-HomoPhe-OH,(S)-5-phenylpentanoic acid, (S)-5-hexenoic acid, (S)-5-phenyl-pentanoicacid, (S)-6-phenyl-5-hexenoic acid,(S)-2-(trifluoromethyl)-β-HomoPhe-OH, (S)-2-(trifluoromethyl)-β-Phe-OH,(S)-2-cyano-β-HomoPhe-OH, (S)-2-methyl-β-Phe-OH,(S)-3,4-dimethoxy-β-Phe-OH, (S)-β-(trifluoromethyl)-β-HomoPhe-OH,(S)-β-(trifluoromethyl)-β-Phe-OH, (S)-3-cyano-β-Phe-OH,(S)-3-methoxy-β-Phe-OH, (S)-3-methyl-β-Phe-OH,(S)-4-(4-pyridyl)-β-HomoAla-OH, (S)-4-(trifluoromethyl)-β-Phe-OH,(S)-4-bromo-β-Phe-OH, (S)-4-chloro-β-HomoPhe-OH, (S)-4-chloro-β-Phe-OH,(S)-4-cyano-β-HomoPhe-OH, (S)-4-cyano-β-Phe-OH, (S)-4-fluoro-β-Phe-OH,(S)-4-iodo-β-HomoPhe-OH, (S)-4-methyl-β-HomoPhe-OH,(S)-4-methyl-β-Phe-OH, (S)-β-Tyr-OH, (S)-γ,γ-diphenyl-β-HomoAla-OH,(S)-2-methyl-β-Homophe-OH, (S)-3,4-difluoro-β-HomoPhe-OH,(S)-β-(trifluoromethyl)-β-HomoPhe-OH, (S)-3-cyano-β-HomoPhe-OH,(S)-3-methyl-β-HomoPhe-OH, (S)-γ,γ-diphenyl-β-HomoAla-OH,3-Amino-3-(3-bromophenyl)propionic acid, and3-Amino-4,4,4-trifluorobutyric acid.

In some embodiments, the fragment comprises 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 amino acids of the wild type protein sequence. In someembodiments, the fragment comprises any of the above-mentioned numbersof amino acids located anywhere within the peptide. Thus, one skilled inthe art understands that a fragment of any of these lengths can bewalked along the length of the peptide, thus providing any fragment ofthe peptide with the same or similar function as the native or wild-typeamino acid sequence.

One of ordinary skill in the art would readily appreciate that theprotecting groups would be removed from the final chemical structure ofthe analog which becomes administered to a subject. One of ordinaryskill would be able to predict the final chemical structure of theanalog by using the protecting groups selectively to create apolypeptide with a desirable chirality or secondary structure. Forinstance, if the analog of the composition is manufactured using(S)-Fmoc-3-methyl-β-HomoPhe-OH, the final yielded product shouldcomprise at least one β-amino acid residue of a3-methyl-β-homophenylalanine.

In some embodiments, the method of manufacturing the analog comprisessynthesizing the analog using at least one, and in some embodiments, aplurality of cyclic amino acid residues. In some embodiments, the VIPanalog of the claimed invention comprises the cyclic amino acidresidues. In some embodiments, the VIP analog of the claimed inventioncomprises at least one disulfide bridge that forms a cyclic chain ofatoms along a side chain of two amino acid residues.

In some embodiments, the VIP analog of the claimed invention comprisesat least 17%β-amino acid residues. In some embodiments, the VIP analogof the claimed invention comprises from about 15% to about 30% β-aminoacid residues. In some embodiments, the VIP analog of the claimedinvention comprises from about 15% to about 30% β-amino acid residueswherein the first ten amino acids of the amino acid sequence are alphaamino acids. In some embodiments, the VIP analog of the claimedinvention comprises from about 16% to about 29% β-amino acid residues.In some embodiments, the VIP analog of the claimed invention comprisesfrom about 17% to about 29% β-amino acid residues. In some embodiments,the VIP analog of the claimed invention comprises from about 18% toabout 29% β-amino acid residues. In some embodiments, the VIP analog ofthe claimed invention comprises from about 19% to about 29% β-amino acidresidues. In some embodiments, the VIP analog of the claimed inventioncomprises from about 20% to about 29% β-amino acid residues.

In some embodiments, at least one of the β³-amino acid residues issubstituted with a residue chosen from the following:(S,S)-trans-2-aminocyclopentanecarboxylic acid ((S,S)-ACPC),(S,R)-trans-2-aminocyclopentanecarboxylic acid ((S,R)-ACPC),(R,S)-trans-2-aminocyclopentanecarboxylic acid ((R,S)-ACPC), or(R,R)-trans-2-aminocyclopentanecarboxylic acid ((R,R)-ACPC) if the aminoacid is non-polar; or pyrrolidine analogue of (S,S)-ACPC, (RS)-ACPC,(S,R)-ACPC, (RR)-ACPC), which is designated APC, if the amino acid isbasic. In some embodiments, at least one of the β³-amino acid residuesis substituted with a residue chosen from the following:(S,S)-trans-2-aminocyclopentanecarboxylic acid ((S,S)-ACPC) if the aminoacid is non-polar; or pyrrolidine analogue of (S,S)-ACPC if the residueis basic.

In some embodiments, the VIP analog of the claimed invention comprisesthe following sequence:

In some embodiments, the VIP analog comprises a cyclic amino acidresidue covalently bonded to one or more contiguous or non-contiguousamino acid sidechain residues via the following synthetic linkingstructures:

In some embodiments, the analog does not comprise a cyclic substituentin its side chain. In some embodiments, the cyclic amino acid residuesare not covalently bonded to one or more contiguous or non-contiguousamino acid sidechain residues via the following synthetic linkingstructures:

In some embodiments, the analogs of the present invention comprise atleast one or a plurality of the following cyclic amino acid residues,some of which being described with a protecting group that becomeseliminated from the analog either during synthesis or when the analog ispurified after synthesis:

-   L-β-HomohydroxyProline hydrochloride-   (1R,2R)-Boc-2-aminocyclohexane carboxylic acid {(1R,2R)-ACHC}-   (1R,2R)-Fmoc-2-aminocyclohexane carboxylic acid {(1R,2R)-ACHC}-   (1R,2S)-Boc-2-aminocyclohexane carboxylic acid {(1R,2S)-ACHC}-   (1R,2S)-Fmoc-2-aminocyclohexane carboxylic acid {(1R,2S)-ACHC}-   (1S,2R)-Boc-2-aminocyclohexane carboxylic acid {(1S,2R)-ACHC}-   (1S,2R)-Fmoc-2-aminocyclohexane carboxylic acid (1S,2R)-ACHC}-   (1S,2S)-Boc-2-aminocyclohexane carboxylic acid {(1S,2S)-ACHC}-   (1S,2S)-Fmoc-2-aminocyclohexane carboxylic acid {(1S,2S)-ACHC}-   (1R,2R)-Boc-2-aminocyclopentane carboxylic acid {(1R,2R)-ACPC}-   (1R,2R)-Fmoc-2-aminocyclopentane carboxylic acid {(1R,2R)-ACPC}-   (1S,2S)-Boc-2-aminocyclopentane carboxylic acid {(1S,2S)-ACPC}-   (1S,2S)-Fmoc-2-aminocyclopentane carboxylic acid {(1S,2S)-ACPC}-   Boc-cis-2-aminocyclopentane carboxylic acid, cis-Acpc-   Fmoc-cis-2-aminocyclopentane carboxylic acid, cis-Acpc-   (R)-Boc-(2-carboxymethyl)-piperidine, (R)-(1-piperidin-2-yl)-acetic    acid-   (R)-Fmoc-(2-carboxymethyl)-piperidine,    (R)-(1-Fmoc-piperidin-2-yl)-acetic acid-   (S)-Boc-(2-carboxymethyl)-piperidine    (S)-(1-Boc-piperidin-2-yl)-acetic acid-   (S)-Fmoc-(2-carboxymethyl)-piperidine    (S)-(1-Fmoc-piperidin-2-yl)-acetic acid-   (R,S)-Boc-2-carboxymorpholine Boc-Cop-   (R,S)-Boc-2-carboxymorpholine Fmoc-Cop-   (R,S)-Boc-nipecotic acid Boc-Nip-   (R,S)-Boc-nipecotic acid Fmoc-Nip-   (R)-Fmoc-nipecotic acid (R)-Fmoc-Nip-   (R)-Fmoc-nipecotic acid (R)-Boc-Nip-   (3S)-Boc-1-pyrrolidine-3-carboxylic acid (3S)-Boc-beta-Pro-OH-   (3S)-Fmoc-1-pyrrolidine-3-carboxylic acid (3S)-Fmoc-beta-Pro-OH

In some embodiments, the analogs of the present invention comprise atleast one or a plurality of non-natural amino acid residues that canmodified by PEGylation. In some embodiments the analogs or fragments ofthe polypeptides related to this invention comprise PEG molecules whichare covalently bound to the side chain of the α, or β amino acids in thepolypeptide. In some embodiments, the polypeptides of this inventioncomprise the PEGylated cyclic amino acid residues or cyclic amino acidside chains. PEG molecule(s) may be covalently attached to any Lys, Cys,K(W) or K(CO(CH₂)₂SH) residue at any position in the analog or fragmentof analog. In some embodiments, the analog or a fragment thereofcomprises a C-terminal extension may comprise one or more Cys residueswhich may be PEGylated. In some embodiment of the invention thepolypeptides or fragments thereof may comprise one or more PEGylatedresidues in either or both sequences.

In some embodiments, the analog or fragment thereof comprises a PEGmolecule covalently attached to one or all of the β-residue within theanalog. In some embodiments, the analog is at least one PEG moleculecovalently attached to a residue in the C-terminal extension of theanalog or fragment thereof. In some embodiments, the analog comprisesmore than one PEG molecule, there may be a combination of Lys, Cys,K(CO(CH₂)₂SH), K(W) and carboxy-terminal amino acid PEGylation. Forexample, if there are two PEG molecules, one may be attached to a Lysresidue and one may be attached to a Cys residue. In some embodiments,the polypeptide comprises one or more covalently bound PEG molecules,wherein at least one of the PEG molecules is branched. In someembodiments, one or more of the PEG molecules are linear. In someembodiments, the composition comprises one or more PEG molecule, whereinthe PEG molecule is between about 200 daltons and about 100,000 daltonsin molecular weight. In some embodiments, the PEG molecule is chosenfrom 10,000, 20,000, 30,000, 40,000, 50,000 and 60,000 daltons. In someembodiments, it is chosen from 20,000, 30,000, 40,000, or 60,000daltons. Where there are two PEG molecules covalently attached to theanalog or fragment thereof, each is 1,000 to 40,000 daltons and, theyhave molecular weights of 20,000 and 20,000 daltons, 10,000 and 30,000daltons, 30,000 and 30,000 daltons, or 20,000 and 40,000 daltons. Insome embodiments mini-PEG s™ are covalently bound to at least oneresidue or side chain of an α, or β-amino acid. In some embodiments, themini-PEG™ is chosen from the following list of products:8-Amino-3,6-Dioxaoctanoic Acid, 11-Amino-3,6,9-Trioxaundecanoic Acid,8-Amino-3,6-Dioxaoctanoic Acid•DCHA, 11-Amino-3,6,9-TrioxaundecanoicAcid•DCHA.

In some embodiments the method of treatment or prevention of a humandisorder depends upon the analog being synthesized. For instance:Peptides for triggering B and T cell activity can be used to treatautoimmune disease, including uveitis, collagen-induced, adjuvant andrheumatoid arthritis, thyroiditis, myasthenia gravis, multiple sclerosisand diabetes. Examples of these peptides are interleukins (referenced inAulitzky, W E; Schuler, M; Peschel, C.; Huber, C.; Interleukins.Clinical pharmacology and therapeutic use. Drugs. 48(5):667-77, November1994) and cytokines (referenced in Peters, M.; Actions of cytokines onthe immune response and viral interactions: an overview. Hepatology.23(4):909-16, April 1996).

Peptides and peptidomimetics that target crucial enzymes, oncogenes oroncogene products, tumor-suppressor genes and their products, growthfactors and their corresponding receptors can be used to treat cancer.Examples of these peptides are described in Unger, C. Current conceptsof treatment in medical oncology: new anticancer drugs. Journal ofCancer Research & Clinical Oncology. 122(4):189-98, 1996.

VIP analogs, agonist analogs and antagonist analogs can be used to treatallergic respiratory diseases, PAH, asthma and allergic rhinitis, andnervous control of reproductive functions.

In some embodiments, the analog is a “bifunctional polymer,” whichrefers to a polymer comprising two discrete functional groups that arecapable of reacting specifically with other moieties (including but notlimited to, amino acid side groups) to form covalent or non-covalentlinkages. A bifunctional linker having one functional group reactivewith a group on a particular biologically active component, and anothergroup reactive with a group on a second biological component, may beused to form a conjugate that includes the first biologically activecomponent, the bifunctional linker and the second biologically activecomponent. Many procedures and linker molecules for attachment ofvarious compounds to peptides are known. See, e.g., European PatentApplication No 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148,4,699,784; 4,680,338; 4,569,789; and 4,589,071 which are incorporated byreference herein. In some embodiments, the analog is a “functionalpolymer” which refers to a polymer comprising two or more discretefunctional groups that are functionally identical and capable ofreacting specifically with other moieties (including but not limited to,amino acid side groups) to form covalent or non-covalent linkages. Afunctional polymer or multi-functional polymer may be any desiredmolecular length or molecular weight, and may be selected to provide aparticular desired spacing or conformation between one or more moleculeslinked to the analog and its binding partner or the analog.

The present invention provides for the use of an antibody or bindingcomposition which specifically binds to a specified analog. in someembodiments the antibody specifically binds the analog derived from amammalian polypeptide, e.g., a polypeptide derived from a primate,human, cat, dog, rat, or mouse. Antibodies can be raised to variousanalogs, including individual, polymorphic, allelic, strain, or speciesvariants, and fragments thereof, both in their naturally occurring(full-length) forms or in their synthetic forms. Additionally,antibodies can be raised to the analogs in their inactive state oractive state. Anti-idiotypic antibodies may also be used.

A number of immunogens may be selected to produce antibodiesspecifically reactive with ligand or receptor proteins. Syntheticanalogs may serva as an immunogen for the production of monoclonal orpolyclonal antibodies. Such antibodies may be used as antagonists oragonists for their targets modulating the disease state associated withthe naturally occurring proteins and analogs listed above. Syntheticpolypeptides of the claimed invention may also be used either in pure orimpure form. Synthetic peptides, made using the appropriate proteinsequences, may also be used as an immunogen for the production ofantibodies. Naturally folded or denatured material can be used, asappropriate, for producing antibodies. Either monoclonal or polyclonalantibodies may be generated, e.g., for subsequent use in immunoassays tomeasure the protein, or for immunopurification methods. Methods ofproducing polyclonal antibodies are well known to those of skill in theart.

Typically, an immunogen, such as a purified analog of the invention, ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to theprotein of interest. For example, when appropriately high titers ofantibody to the immunogen are obtained, usually after repeatedimmunizations, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be performed if desired. See, e.g., Harlowand Lane; or Coligan. Immunization can also be performed through othermethods, e.g., DNA vector immunization. See, e.g., Wang, et al. (1997)Virology 228:278-284.

Monoclonal antibodies may be obtained by various techniques familiar toresearchers skilled in the art. Typically, spleen cells from an animalimmunized with a desired analog are immortalized, commonly by fusionwith a myeloma cell. See, Kohler and Milstein (1976) Eur. J. Immunol.6:511-519. Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodsknown in the art. See, e.g., Doyle, et al. (eds. 1994 and periodicsupplements) Cell and Tissue Culture: Laboratory Procedures, John Wileyand Sons, New York, N.Y. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according, e.g., to the general protocol outlined by Huse,et al. (1989) Science 246:1275-1281.

Antibodies or binding compositions, including binding fragments, singlechain antibodies, F_(v), F_(ab), single domain V_(H), disulfide-bridgedF_(v), single-chain F_(v) or F(_(ab)′)₂ fragments of antibodies,diabodies, and triabodies against predetermined fragments of the analogscan be raised by immunization of animals with analogs or conjugates ofanalogs or receptor proteins with carrier proteins. Monoclonalantibodies are prepared from cells VIPg the desired antibody. Theseantibodies can be screened for binding to analogs described herein.These monoclonal antibodies will usually bind with at least a K_(D) ofabout 1 mM, usually at least about 300 μM, typically at least about 10μM, at least about 30 μM, at least about 10 μM, and at least about 3 μMor more. These antibodies can be screened for binding to the naturallyoccurring polypeptides upon which the analogs are derived.

In some instances, it is desirable to prepare monoclonal antibodies(mAbs) from various mammalian hosts, such as mice, rodents, primates,humans, etc. Description of techniques for preparing such monoclonalantibodies may be found in, e.g., Stites, et al. (eds.) Basic andClinical Immunology, 4th ed., Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) Antibodies:A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies:Principles and Practice, 2nd ed., Academic Press, New York, N.Y.; andparticularly in Kohler and Milstein (1975) Nature 256:495-497, whichdiscusses one method of generating monoclonal antibodies Summarizedbriefly, this method involves injecting an animal with an analogdescribed herein. The animal is then sacrificed and cells taken from itsspleen, which are then fused with myeloma cells. The result is a hybridcell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the analog. In thismanner, the individual antibody species obtained are the products ofimmortalized and cloned single B cells from the immune animal generatedin response to a specific site recognized on the immunogenic substance.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse, et al. (1989) Science246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. Thepolypeptides and antibodies of the present invention may be used with orwithout modification, including chimeric or humanized antibodies.Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties,magnetic particles, and the like. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinantimmunoglobulins may be produced, see, Cabilly, U.S. Pat. No. 4,816,567;and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; ormade in transgenic mice, see Mendez, et al. (1997) Nature Genetics15:146-156; also see Abgenix and Medarex technologies.

The instant invention is related to pharmaceutical compositions of theinstant invention or the pharmaceutical acceptable salts derivedtherefrom that comprise analogs that comprise isotopes. In someembodiments, the compositions of the claimed invention may contain anyisotope described in Cyr and Pearson (Stabilization ofradiopharmaceutical compositions using hydrophilic thioethers andhydrophilic 6-hydroxy chromans. Cyr, John E.; Pearson, Daniel A.(Diatide, Inc., USA). PCT Int. Appl. (2002), WO 200260491 A2 20020808),which is herein incorporated by reference. In some embodiments thecompositions of the invention comprise analog that comprise one or moreof the following isotopes: ¹²⁵I, ¹³¹I, ²¹¹At, ⁴⁷Sc, ⁶⁷Cu, ⁷²Ga, ⁹⁰Y,¹⁵³Sm, ¹⁵⁹Gd, ¹⁶⁵Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ⁶⁸Ga, ⁹⁹Tc,¹¹¹In, ¹²³I, and ³H.

The pharmaceutical compositions of the instant invention or thepharmaceutical acceptable salts derived therefrom may be in a liquid orsolid dosage form. Such compositions may include any type of dosage formsuch as tablets, capsules, powders, liquid formulations, delayed orsustained release, patches, snuffs, nasal sprays and the like. Theformulations may additionally include other ingredients such as dyes,preservatives, buffers and anti-oxidants, for example. The physical formand content of the pharmaceutical formulations contemplated areconventional preparations that can be formulated by those skilled in thepharmaceutical formulation field and are based on well establishedprinciples and compositions described in, for example, Remington: TheScience and Practice of Pharmacy, 19th Edition, 1995; BritishPharmacopoeia 2000, each of which is incorporated herein by reference.The compositions of the present invention may also include other activeagents useful in the treatment of cardiovascular conditions. Solid formscan be prepared according to any means suitable in the art. For example,capsules are prepared by mixing the analog composition with a suitablediluent and filling the proper amount of the mixture in capsules.Tablets are prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants and disintegrators as well as the compound.Diluents, but are not limited to, include various types of starch,cellulose, crystalline cellulose, microcrystalline cellulose, lactose,fructose, sucrose, mannitol or other sugar alcohols, kaolin, calciumphosphate or sulfate, inorganic salts such as sodium chloride andpowdered sugar. Powdered cellulose derivatives are also useful.Non-limiting examples of tablet binders include, but are not limited to,starches, gelatin and sugars such as lactose, fructose, glucose and thelike. Natural and synthetic gums are also convenient, including, but arenot limited to, acacia, alginates, methylcellulose, polyvinylpyrrolidoneand the like. Polyethylene glycol, ethylcellulose and waxes can alsoserve as binders.

A lubricant can be used in a tablet formulation to prevent the tabletand punches from sticking in the die. The lubricant include, but are notlimited to, such slippery solids as talc, magnesium and calciumstearate, stearic acid and hydrogenated vegetable oils.

Tablets can be coated with sugar as a flavor and sealant, or withfilm-forming protecting agents to modify the dissolution properties ofthe tablet. The compounds may also be formulated as chewable tablets, byusing large amounts of pleasant-tasting substances such as mannitol inthe formulation, as is now well-established in the art.

Also contemplated are liquid formulations and solid form preparationswhich are intended to be converted, shortly before use, to liquid formpreparations. Such liquid forms include, but are not limited to,solutions, suspensions, syrups, slurries, and emulsions. Liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats or oils); emulsifyingagents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almondoil, oily esters, or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thesepreparations may contain, in addition to the active agent, colorants,flavors, stabilizers, buffers, artificial and natural sweeteners,dispersants, thickeners, solubilizing agents, and the like. Thecompositions may be in powder form for constitution with a suitablevehicle such as sterile water, saline solution, or alcohol, before use.Preparations may also contain mucosal enhancers. In some embodiments,the oral transmucosal solid dosage further comprises a permeationenhancer. In some embodiments, the permeation enhancer is chosen from: abile salt, sodium dodecyl sulfate, dimethyl sulfoxide, sodium laurylsulfate, a derivative of a saturated or a unsaturated fatty acid, asurfactant, a bile salt analog, and a derivative of a bile salt. In someembodiments the oral transmucosal dosage form is chosen from: a chewinggum, a patch, a lozenge, a lozenge-on-a-handle, a tablet, a troche, apastille, a sachet, a sublingual tablet, and a rapid disintegratingtablet. In some embodiments, the oral transmucosal solid dosage form ofwherein the composition further comprises at least one flavoring agent,artificial coloring, sweetener, lubricating agent, disintegration agent,lubricating agent, diluent, base, or buffering agent. In someembodiments, the oral transmucosal solid dosage form further comprises asustained release agent. The invention is directed to an oraltransmucosal solid dosage form comprising from wherein the concentrationof analog is from about 0.01% to about 90% of the dry matter weight ofthe composition.

Solid dosage forms such as lozenges and tablets may also be used fororal transmucosal delivery of pharmaceuticals. For example,nitroglycerin sublingual tablets have been on the market for many years.The sublingual tablets are designed to deliver small amounts of thepotent nitroglycerin, which is almost immediately dissolved andabsorbed. On the other hand, most lozenges or tablets are typicallydesigned to dissolve in the mouth over a period of at least severalminutes which allows extended dissolution of the lozenge and absorptionof the drug.

Administration of lozenges or sublingual tablets generally utilize an“open” delivery system, in which the drug delivery conditions areinfluenced by the conditions of the surrounding environment, such asrate of saliva secretion, pH of the saliva, or other conditions beyondthe control of the formulation.

A lozenge-on-a-handle (similar to a lollipop) is another dosage formsuitable for transmucosal drug delivery. In addition to beingnon-invasive and providing a particularly easy method of delivery, thelozenge-on-a-handle (or lozenge with an integrated oral transmucosalapplicator) dosage form allows a patient or caregiver to move the dosageform in and out of the mouth to titrate the dose. This practice iscalled dose-to-effect, in which a patient or caregiver controls theadministration of the dose until the expected therapeutic effect isachieved. This is particularly important for certain symptoms, such aspain, nausea, motion sickness, and premedication prior to anesthesiabecause each patient needs a different amount of medication to treatthese symptoms. For these types of treatments, the patient is the onlyone who knows how much medication is enough. Once the appropriate amountof drug is delivered, the patient or caregiver can remove thelozenge-on-a-handle, thus, stopping delivery of the drug. This featureis especially important for particularly potent drugs, which may presenta significant advantage of terminating drug administration once thedesired effect is achieved.

As used herein, the term “oral transmucosal delivery” (OTD) refers tothe delivery of a pharmaceutical agent across a mucous membrane in theoral cavity, pharyngeal cavity, or esophagus, and may be contrasted, forexample, with traditional oral delivery, in which absorption of the drugoccurs in the intestines. Accordingly, routes of administration in whichthe pharmaceutical agent is absorbed through the buccal, sublingual,gingival, pharyngeal, and/or esophageal mucosa are all encompassedwithin “oral transmucosal delivery,” as that term is used herein. Oraltransmucosal delivery involves the administration of an oraltransmucosal solid dosage form to the oral cavity of a patient, which isheld in the oral cavity and dissolved, thereby releasing thepharmaceutical agent for oral transmucosal delivery. Of course, as thesolid dosage form dissolves in the oral cavity, some of the salivacontaining the pharmaceutical agent may be swallowed, and a portion ofthe drug may ultimately be absorbed from the intestines.

The compositions of the invention can be administered in a sustainedrelease composition, such as those described in, for example, U.S. Pat.No. 5,672,659 and U.S. Pat. No. 5,595,760, and herein incorporate byreference. The use of immediate or sustained release compositionsdepends on the type of condition being treated.

The pharmaceutical compositions of the instant invention or thepharmaceutical acceptable salts derived therefrom may be in a dosageamount in an effective amount for inducing or increasing the naturallyoccurring biological activity of the wild-type polypeptide upon whichthe analog is derived. The pharmaceutical compositions of the instantinvention or the pharmaceutical acceptable salts derived therefrom maybe in a dosage amount in an effective amount for inducing or increasingthe naturally occurring biological activity of the wild-type VIPpolypeptide upon which the analog is derived. The pharmaceuticalcompositions of the instant invention or the pharmaceutical acceptablesalts derived therefrom may be in a dosage amount in an effective amountfor increasing the half-life of the composition when administered to ahuman being or other subject. In some embodiments the VIP analog is VIP.

The present invention also encompasses methods of using the compositionscomprising a VIP analog. Any of these methods may involve theadministration of a pharmaceutical composition comprising a VIP analogwherein the VIP analog is in a therapeutically effective dose. Any ofthese methods may involve the administration of a pharmaceuticalcomposition comprising a VIP analog wherein the VIP analog is selectivefor VPAC1, VPAC2, PAC1, VIPR1, or VIPR2 at a magnitude between 1-100time greater than the other VIP receptors disclosed herein. Thecomposition comprising an analog of the invention produces a broad rangeof activities, depending on the dosage administered. The presentinvention encompasses methods of treating or preventing pulmonaryhypertension, primary arterial hypertension, pulmonary hypertensionassociated to post-ventricular septal defect, idiopathic pulmonaryfibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small lung cellcancer, exocrine pancreatic tumors, colorectal carcinoma, gastriccarcinoma, hepatocellular carcinoma, esophageal carcinoma, renal cellcarcinoma, prostate carcinoma, urinary bladder carcinoma, livercarcinoma, ductal pancreatic cancer, breast carcinoma, ovariancarcinoma, non-hodgkin's lymphoma, meningioma, GEP tumors(differentiated and undifferentiated), pituitary adenoma, endometrialcancer, astrocytoma, giloblastoma, non-small cell lung cancer,pancreatic cancer, melanoma, renal cancer, neuroblastoma, leukima,prostate cancer, autoimmune disease, inflammatory disease, sepsis,Hirschsprung's Disease, sexual dysfunction, erectile dysfunction,Parkinson's disease, Alzheimer's disease, circadian rhythm dysfunction,pain, colorectal cancer, hepatocellular cancer, elevated blood pressurelevels, elevated blood glucose levels, hyperglycemia, diabetes, insulinresistance, metabolic acidosis, obesity, Type I diabetes, Type IIdiabetes Multiple Sclerosis, osteoporosis, Sjogren's syndrome,pancreatitis, uveoretinitis, osteoporosis, female sexual dysfunctioncomprising administering to at least one patient in need thereof, mammalin need thereof or human in need thereof a composition or pharmaceuticalcomposition comprising a VIP family analog in a therapeuticallyeffective amount. The compositions of the invention may also be used atlower doses in order to prevent pulmonary hypertension, primary arterialhypertension, pulmonary hypertension associated to post-ventricularseptal defect, idiopathic pulmonary fibrosis, idiopathic pulmonaryarterial hypertension, CREST syndrome—Calcinosis; Raynaud's disease;loss of muscle control of the Esophagus; Sclerodactyly; Telangiectasia,Acute respiratory distress, congestive heart failure, chronic obstructedpulmonary disorder, asthma, chronic obstructive pulmonary disease,sarcoidosis, small lung cell cancer, autoimmune disease, inflammatorydisease, sepsis, Hirschsprung's Disease, sexual dysfunction, erectiledysfunction, Parkinson's disease, Alzheimer's disease, circadian rhythmdysfunction, pain, colorectal cancer, hepatocellular cancer, elevatedblood pressure levels, elevated blood glucose levels, hyperglycemia,diabetes, insulin resistance, metabolic acidosis, obesity, Type Idiabetes, Type II diabetes Multiple Sclerosis, osteoporosis, Sjogren'ssyndrome, pancreatitis, uveoretinitis, osteoporosis, female sexualdysfunction in a subject in need thereof. The compositions of theinvention may also be used to prevent pulmonary hypertension, primaryarterial hypertension, pulmonary hypertension associated topost-ventricular septal defect, idiopathic pulmonary fibrosis,idiopathic pulmonary arterial hypertension, CREST syndrome—Calcinosis;Raynaud's disease; loss of muscle control of the Esophagus;Sclerodactyly; Telangiectasia, Acute respiratory distress, congestiveheart failure, chronic obstructed pulmonary disorder, asthma, chronicobstructive pulmonary disease, sarcoidosis, small lung cell cancer,autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, hyperglycemia, diabetes, insulin resistance,metabolic acidosis, obesity, Type I diabetes, Type II diabetes MultipleSclerosis, osteoporosis, Sjogren's syndrome, pancreatitis,uveoretinitis, osteoporosis, female sexual dysfunction in a subjectsusceptible to those indications. In some embodiments, the method ofprevention comprising administering the composition or pharmaceuticalcompositions of the invention after the subject is tested forsusceptibility or genetic propensity for developing the disease,indication or disorder.

The pharmaceutical composition comprising a pharmaceutically acceptablecarrier/diluent and an analog comprising an α-amino acid and at leastone β-amino acid may be formulated by one having ordinary skill in theart with compositions selected depending upon the chosen mode ofadministration. Suitable pharmaceutical carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, A. Osol, astandard reference text in this field, which is incorporated herein inits entirety.

For parenteral administration, analog can be, for example, formulated asa solution, suspension, emulsion or lyophilized powder in associationwith a pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes and nonaqueous vehicles such as fixedoils may also be used. The vehicle or lyophilized powder may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of analog in 0.9% sodium chloridesolution.

The present invention relates to routes of administration includeintramuscular, sublingual, intravenous, intraperitoneal, intrathecal,intravaginal, intraurethral, intradermal, intrabuccal, via inhalation,via nebulizer and via subcutaneous injection. Alternatively, thepharmaceutical composition may be introduced by various means into cellsthat are removed from the individual. Such means include, for example,microprojectile bombardment and liposome or other nanoparticle device.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In solid dosage forms, the analogs aregenerally admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, starch, or other generally regarded assafe (GRAS) additives. Such dosage forms can also comprise, as is normalpractice, an additional substance other than an inert diluent, e.g.,lubricating agent such as magnesium state. With capsules, tablets, andpills, the dosage forms may also comprise a buffering agent. Tablets andpills can additionally be prepared with enteric coatings, or in acontrolled release form, using techniques know in the art.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions and syrups, with theelixirs containing an inert diluent commonly used in the art, such aswater. These compositions can also include one or more adjuvants, suchas wetting agent, an emulsifying agent, a suspending agent, a sweeteningagent, a flavoring agent or a perfuming agent.

In another embodiment of the invention the composition of the inventionis used to treat a patient suffering from, or susceptible to, pulmonaryhypertension, primary arterial hypertension, pulmonary hypertensionassociated to post-ventricular septal defect, idiopathic pulmonaryfibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small lung cellcancer, autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, hyperglycemia, diabetes, insulin resistance,metabolic acidosis, obesity, Type I diabetes, Type II diabetes MultipleSclerosis, osteoporosis, Sjogren's syndrome, pancreatitis,uveoretinitis, osteoporosis, female sexual dysfunction due toadministration of a medication that causes onset of or exacerbatessymptoms of pulmonary hypertension, primary arterial hypertension,pulmonary hypertension associated to post-ventricular septal defect,idiopathic pulmonary fibrosis, idiopathic pulmonary arterialhypertension, CREST syndrome—Calcinosis; Raynaud's disease; loss ofmuscle control of the Esophagus; Sclerodactyly; Telangiectasia, Acuterespiratory distress, congestive heart failure, chronic obstructedpulmonary disorder, asthma, chronic obstructive pulmonary disease,sarcoidosis, small lung cell cancer, autoimmune disease, inflammatorydisease, sepsis, Hirschsprung's Disease, sexual dysfunction, erectiledysfunction, Parkinson's disease, Alzheimer's disease, circadian rhythmdysfunction, pain, colorectal cancer, hepatocellular cancer, elevatedblood pressure levels, elevated blood glucose levels, hyperglycemia,diabetes, insulin resistance, metabolic acidosis, obesity, Type Idiabetes, Type II diabetes Multiple Sclerosis, osteoporosis, Sjogren'ssyndrome, pancreatitis, uveoretinitis, osteoporosis, female sexualdysfunction in a subject. In some embodiments, the invention relates tocompositions comprising a VIP family analog for treatment or preventionof pulmonary hypertension, primary arterial hypertension, pulmonaryhypertension associated to post-ventricular septal defect, idiopathicpulmonary fibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small lung cellcancer, autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, hyperglycemia, diabetes, insulin resistance,metabolic acidosis, obesity, Type I diabetes, Type II diabetes MultipleSclerosis, osteoporosis, Sjogren's syndrome, pancreatitis,uveoretinitis, osteoporosis, female sexual dysfunction in a subject inneed thereof, wherein said analog comprises an α-amino acid and at leastone β-amino acid. In some embodiments, the VIP family analog of theinvention comprises an analog of VIP.

One of skill in the art will recognize that the appropriate dosage ofthe compositions and pharmaceutical compositions may vary depending onthe individual being treated and the purpose. For example, the age, bodyweight, and medical history of the individual patient may affect thetherapeutic efficacy of the therapy. Further, a lower dosage of thecomposition may be needed to produce a transient cessation of symptoms,while a larger dose may be needed to produce a complete cessation ofsymptoms associated with the disease, disorder, or indication. Acompetent physician can consider these factors and adjust the dosingregimen to ensure the dose is achieving the desired therapeutic outcomewithout undue experimentation. It is also noted that the clinicianand/or treating physician will know how and when to interrupt, adjust,and/or terminate therapy in conjunction with individual patientresponse. Dosages may also depend on the strength of the particularanalog chosen for the pharmaceutical composition.

The dose of the composition or pharmaceutical compositions may vary. Thedose of the composition may be once per day. In some embodiments,multiple doses may be administered to the subject per day. In someembodiments, the total dosage is administered in at least twoapplication periods. In some embodiments, the period can be an hour, aday, a month, a year, a week, or a two-week period. In an additionalembodiment of the invention, the total dosage is administered in two ormore separate application periods, or separate doses.

In some embodiments, subjects can be administered the composition inwhich the composition is provided in a daily dose range of about 0.0001mg/kg to about 5000 mg/kg of the weight of the subject. The doseadministered to the subject can also be measured in terms of totalamount of analog administered per day. In some embodiments, a subject isadministered from about 0.001 to about 3000 milligrams of analog perday. In some embodiments, a subject is administered up to about 2000milligrams of analog per day. In some embodiments, a subject isadministered up to about 1800 milligrams of analog per day. In someembodiments, a subject is administered up to about 1600 milligrams ofanalog per day. In some embodiments, a subject is administered up toabout 1400 milligrams of analog per day. In some embodiments, a subjectis administered up to about 1200 milligrams of analog per day. In someembodiments, a subject is administered up to about 1000 milligrams ofanalog per day. In some embodiments, a subject is administered up toabout 800 milligrams of analog per day. In some embodiments, a subjectis administered from about 0.001 milligrams to about 700 milligrams ofanalog per dose. In some embodiments, a subject is administered up toabout 700 milligrams of analog per dose. In some embodiments, a subjectis administered up to about 600 milligrams of analog per dose. In someembodiments, a subject is administered up to about 500 milligrams ofanalog per dose. In some embodiments, a subject is administered up toabout 400 milligrams of analog per dose. In some embodiments, a subjectis administered up to about 300 milligrams of VIP analog per dose. Insome embodiments, a subject is administered up to about 200 milligramsof analog per dose. In some embodiments, a subject is administered up toabout 100 milligrams of analog per dose. In some embodiments, a subjectis administered up to about 50 milligrams of analog per dose.

In some embodiments, subjects can be administered the composition inwhich the composition comprising a VIP analog or pharmaceuticallyacceptable salt thereof is administered in a daily dose range of about0.0001 mg/kg to about 5000 mg/kg of the weight of the subject. In someembodiments, the composition comprising a VIP analog or pharmaceuticallyacceptable salt thereof is administered in a daily dosage of up about450 mg/kg of the weight of the subject. In some embodiments, thecomposition comprising a VIP analog or pharmaceutically acceptable saltthereof is administered in a daily dosage of up about 400 mg/kg of theweight of the subject. In some embodiments, the composition comprising aVIP analog or pharmaceutically acceptable salt thereof is administeredin a daily dosage of up about 350 mg/kg of the weight of the subject. Insome embodiments, the composition comprising a VIP analog orpharmaceutically acceptable salt thereof is administered in a dailydosage of up about 300 mg/kg of the weight of the subject. In someembodiments, the composition comprising a VIP analog or pharmaceuticallyacceptable salt thereof is administered in a daily dosage of up about250 mg/kg of the weight of the subject. In some embodiments, thecomposition comprising a VIP analog or pharmaceutically acceptable saltthereof is administered in a daily dosage of up about 200 mg/kg of theweight of the subject. In some embodiments, the composition comprising aVIP analog or pharmaceutically acceptable salt thereof is administeredin a daily dosage of up about 150 mg/kg of the weight of the subject. Insome embodiments, the composition comprising a VIP analog orpharmaceutically acceptable salt thereof is administered in a dailydosage of up about 100 mg/kg of the weight of the subject. In someembodiments, the composition comprising a VIP analog or pharmaceuticallyacceptable salt thereof is administered in a daily dosage of up about 50mg/kg of the weight of the subject. In some embodiments, the compositioncomprising a VIP analog or pharmaceutically acceptable salt thereof isadministered in a daily dosage of up about 25 mg/kg of the weight of thesubject.

In some embodiments, the composition comprising a VIP analog orpharmaceutically acceptable salt thereof is administered in a dailydosage of up about 10 mg/kg of the weight of the subject. In someembodiments, the composition comprising a VIP analog or pharmaceuticallyacceptable salt thereof is administered in a daily dosage of up about 5mg/kg of the weight of the subject. In some embodiments, the compositioncomprising a VIP analog or pharmaceutically acceptable salt thereof isadministered in a daily dosage of up about 1 mg/kg of the weight of thesubject. In some embodiments, the composition comprising a VIP analog orpharmaceutically acceptable salt thereof is administered in a dailydosage of up about 0.1 mg/kg of the weight of the subject. In someembodiments, the composition comprising a VIP analog or pharmaceuticallyacceptable salt thereof is administered in a daily dosage of up about0.01 mg/kg of the weight of the subject. In some embodiments, thecomposition comprising a VIP analog or pharmaceutically acceptable saltthereof is administered in a daily dosage of up about 0.001 mg/kg of theweight of the subject. The dose administered to the subject can also bemeasured in terms of total amount of VIP analog administered per day.

In some embodiments, a subject in need thereof is administered fromabout 1 ng to about 500 μg of analog or pharmaceutically salt thereofper day. In some embodiments, a subject in need thereof is administeredfrom about 1 ng to about 10 ng of analog or pharmaceutically saltthereof per day. In some embodiments, a subject in need thereof isadministered from about 10 ng to about 20 ng of analog orpharmaceutically salt thereof per day. In some embodiments, a subject inneed thereof is administered from about 10 ng to about 100 ng of analogor pharmaceutically salt thereof per day. In some embodiments, a subjectin need thereof is administered from about 100 ng to about 200 ng ofanalog or pharmaceutically salt thereof per day. In some embodiments, asubject in need thereof is administered from about 200 ng to about 300ng of analog or pharmaceutically salt thereof per day. In someembodiments, a subject in need thereof is administered from about 300 ngto about 400 ng of analog or pharmaceutically salt thereof per day. Insome embodiments, a subject in need thereof is administered from about400 ng to about 500 ng of analog or pharmaceutically salt thereof perday. In some embodiments, a subject in need thereof is administered fromabout 500 ng to about 600 ng of analog or pharmaceutically salt thereofper day. In some embodiments, a subject in need thereof is administeredfrom about 600 ng to about 700 ng of analog or pharmaceutically saltthereof per day. In some embodiments, a subject in need thereof isadministered from about 800 ng to about 900 ng of analog orpharmaceutically salt thereof per day. In some embodiments, a subject inneed thereof is administered from about 900 ng to about 1 μg of analogor pharmaceutically salt thereof per day. In some embodiments, a subjectin need thereof is administered from about 1 μg to about 100 μg ofanalog or pharmaceutically salt thereof per day. In some embodiments, asubject in need thereof is administered from about 100 μg to about 200μg of analog or pharmaceutically salt thereof per day. In someembodiments, a subject in need thereof is administered from about 200 μgto about 300 μg of analog or pharmaceutically salt thereof per day. Insome embodiments, a subject in need thereof is administered from about300 μg to about 400 μg of analog or pharmaceutically salt thereof perday. In some embodiments, a subject in need thereof is administered fromabout 400 μg to about 500 μg of analog or pharmaceutically salt thereofper day. In some embodiments, a subject in need thereof is administeredfrom about 500 μg to about 600 μg of analog or pharmaceutically saltthereof per day. In some embodiments, a subject in need thereof isadministered from about 600 μg to about 700 μg of analog orpharmaceutically salt thereof per day. In some embodiments, a subject inneed thereof is administered from about 800 μg to about 900 μg of analogor pharmaceutically salt thereof per day. In some embodiments, a subjectin need thereof is administered from about 900 μg to about 1 mg ofanalog or pharmaceutically salt thereof per day.

In some embodiments, a subject in need thereof is administered fromabout 0.0001 to about 3000 milligrams of VIP analog or pharmaceuticallysalt thereof per day. In some embodiments, a subject is administered upto about 2000 milligrams of VIP analog or pharmaceutically salt thereofday. In some embodiments, a subject is administered up to about 1800milligrams of VIP analog or pharmaceutically salt thereof per day. Insome embodiments, a subject is administered up to about 1600 milligramsof VIP analog or pharmaceutically salt thereof per day. In someembodiments, a subject is administered up to about 1400 milligrams ofVIP analog or pharmaceutically salt thereof per day. In someembodiments, a subject is administered up to about 1200 milligrams ofVIP analog or pharmaceutically salt thereof per day. In someembodiments, a subject is administered up to about 1000 milligrams ofVIP analog or pharmaceutically salt thereof per day. In someembodiments, a subject is administered up to about 800 milligrams of VIPanalog or pharmaceutically salt thereof per day. In some embodiments, asubject is administered from about 0.0001 milligrams to about 700milligrams of VIP analog or pharmaceutically salt thereof per dose. Insome embodiments, a subject is administered up to about 700 milligramsof VIP analog or pharmaceutically salt thereof per dose. In someembodiments, a subject is administered up to about 600 milligrams of VIPanalog or pharmaceutically salt thereof per dose. In some embodiments, asubject is administered up to about 500 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 400 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 300 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 200 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 100 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 50 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 25 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 15 milligrams of VIP analog orpharmaceutically salt thereof per dose.

In some embodiments, a subject is administered up to about 10 milligramsof VIP analog or pharmaceutically salt thereof per dose. In someembodiments, a subject is administered up to about 5 milligrams of VIPanalog or pharmaceutically salt thereof per dose. In some embodiments, asubject is administered up to about 1 milligram of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 0.1 milligrams of VIP analog orpharmaceutically salt thereof per dose. In some embodiments, a subjectis administered up to about 0.001 milligrams of VIP analog orpharmaceutically salt thereof per dose.

The dose administered to the subject can also be measured in terms oftotal amount of VIP analog or pharmaceutically salt thereof administeredper ounce of liquid prepared. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 2.5 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 2.25 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 2.25 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 2.0 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.9 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.8 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.7 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.6 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.5 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.4 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.3 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.2 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.1 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 1.0 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.9 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.8 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.7 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.6 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.5 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.4 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.3 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.2 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.1 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.01 gramsper ounce of solution. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.001 gramsper ounce of solution prepared. In some embodiments, the VIP analog orpharmaceutically salt thereof is at a concentration of about 0.0001grams per ounce of solution prepared. In some embodiments, the VIPanalog or pharmaceutically salt thereof is at a concentration of about0.00001 grams per ounce of solution prepared. In some embodiments, theVIP analog or pharmaceutically salt thereof is at a concentration ofabout 0.000001 grams per ounce of solution prepared.

Dosage may be measured in terms of mass amount of analog per liter ofliquid formulation prepared. One skilled in the art can increase ordecrease the concentration of the analog in the dose depending upon thestrength of biological activity desired to treat or prevent anyabove-mentioned disorders associated with the treatment of subjects inneed thereof. For instance, one embodiment of the invention can includeup to 0.00001 grams of analog per 5 mL of liquid formulation and up toabout 10 grams of analog per 5 mL of liquid formulation.

In some embodiments the pharmaceutical compositions of the claimedinvention comprise at least one other active agent. In some embodimentsthe active agent is covalently linked to the VIP analog disclosed hereinoptionally by a protease cleavable linker (including by not limited toPro-Pro or Cituline-Valine di-α-amino acid linkers). In someembodiments, at least one active agent is a chemotherapeutic agent. Insome embodiments, the at least one chemotherapeutic agent is chosenfrom: Mitotic inhibitors, including Dolastatin and auristatin basedcompounds such as Dola-10 or Dola-15 or MMAE, Taxanes includingpaclitaxel, Maytansinoid including Maytansine like based compounds,Alkaloids including vincristine like compounds; Antibiotic basedcompounds including enediyne antibiotics such as calicheamicins andantharcyclines such as Doxorubicin; Alkylating agents including agentsthat modify DNA such as cisplatin or carboplatin; Antimetabolites, suchas methotrexate based compounds and Topoisomerase Inhibitor basedcompounds including Camptothecin and Etoposide or a combination thereof.

In some embodiments, the active agent is a vasoactive agent. In someembodiments the vasoactive agent is chosen from the naturally occurringprostaglandins prostaglandin E0 (PGE0, also referred to13,14-dihydro-PGE1; hereinafter, the abbreviation “PG” is used for“prostaglandin”), PGE1, 19-hydroxy-PGE1, PGE2, 19-hydroxy-PGE2, PGA1,19-hydroxy-PGA1, PGA2, 19-hydroxy-PGA2, PGB1, 19-hydroxy-PGB1, PGB2,19-hydroxy-PGB2, PGB3, PGD2, PGF1α, PGF2α(dinoprost), PGE3, PGF3α, PGI2(prostacyclin), and combinations thereof. PGE0, PGE1, PGE2, and thehydrolyzable lower alkyl esters thereof (e.g., the methyl, ethyl andisopropyl esters) are, however, particularly suitable. Other suitableprostaglandins are exemplified, without limitation, by arboprostil,carbaprostacyclin, carboprost tromethamine, dinoprost tromethamine,dinoprostone, enprostil, iloprost, lipoprost, gemeprost, metenoprost,sulprostone, tiaprost, viprostil (CL 115,347), viprostil methyl ester,16,16-dimethyl-Δ2-PGE1 methyl ester, 15-deoxy-16-hydroxy-16-methyl-PGE1methyl ester (misoprostol), 16,16-dimethyl-PGE1,11-deoxy-15-methyl-PGE1,16-methyl-18,18,19,19-tetrahydrocarbacyclin,16(RS)-15-deoxy-16-hydroxy-16-methyl-PGE1 methyl ester,(+)-4,5-didehydro-16-phenoxy-α-tetranor-PGE2 methyl ester,11-deoxy-11α,16,16-trimethyl-PGE2,(+)-11α,16α,16β-dihydroxy-1-(hydroxymethyl)-16-methyl-trans-prostene,9-chloro-16,16-dimethyl-PGE2, 16,16-dimethyl-PGE2,15(S)-15-methyl-PGE2,9-deoxy-9-methylene-16,16-dimethyl-PGE2, potassiumsalt, 19(R)-hydroxy-PGE2, and 11-deoxy-16,16-dimethyl-PGE2. Additionalvasoactive agents useful as secondary active agents herein includeendothelin-derived relaxation factors (“EDRFs”) such as nitric oxidereleasing agents, e.g., sodium nitroprusside and diazenium diolates, or“NONOates.” NONOates include, but are not limited to,(Z)-1-{N-methyl-N-{6-(N-methyl-ammoniohexyl)amino}}diazen-1-ium-1,2-diolate(“MAHMA/NO”),(Z)-1-{N-(3-ammoniopropyl)-N-(n-propyl)amino}-diazen-1-ium-1,2-diolate(“PAPA/NO”),(Z)-1-{N-{3-aminopropyl}-N-{4-(3-aminopropylammonio)butyl}amino}diazen-1-ium-1,2-diolate(spermine NONOate or “SPER/NO”) and sodium(Z)-1-(N,N-diethylamino)-diazen-1-ium-1,2-diolate (diethylamine NONOateor “DEA/NO”) and derivatives thereof). Still other vasoactive agentsinclude vasoactive intestinal polypeptide analogs and derivativesthereof (particularly derivatives in the form of hydrolyzable loweralkyl esters), smooth muscle relaxants, leukotriene inhibitors, calciumchannel blockers, P2-adrenergic agonists, angiotensin-converting enzyme(“ACE”) inhibitors, angiotensin II receptor antagonists, andphosphodiesterase inhibitors. Still other suitable vasoactive agentsinclude, but are not limited to: nitrates and like compounds such asnitroglycerin, isosorbide dinitrate, erythrityl tetranitrate, amylnitrate, molsidomine, linsidomine chlorhydrate (“SIN-1”),S-nitroso-N-acetyl-d,l-penicillamine (“SNAP”) andS-nitroso-N-glutathione (“SNO-GLU”); long and short acting α-blockerssuch as phenoxybenzamine, dibenamine, doxazosin, terazosin,phentolamine, tolazoline, prazosin, trimazosin, alfuzosin, tamsulosinand indoramin; ergot alkaloids such as ergotamine and ergotamineanalogs, e.g., acetergamine, brazergoline, bromerguride, cianergoline,delorgotrile, disulergine, ergonovine maleate, ergotamine tartrate,etisulergine, lergotrile, lysergide, mesulergine, metergoline,metergotamine, nicergoline, pergolide, propisergide, proterguride andterguride; antihypertensive agents such as diazoxide, hydralazine andminoxidil; nimodepine; pinacidil; cyclandelate; dipyridamole;isoxsuprine; chlorpromazine; haloperidol; yohimbine; and trazodone.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is an inhibitor of rho kinase, an enzymebelonging to the rhoA/rho associated kinase pathway, which regulates thestate of phosphorylation of myosin phosphatase, in turn leading to thecontrol of smooth muscle contraction. One example of a suitable rhokinase inhibitor has the following structural formula and is identifiedas Y-27632. Other suitable rho kinase inhibitors are disclosed, forexample, in U.S. Pat. No. 6,218,410, which is herein incorporated byreference.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that are peptide analogs ofα-melanocyte-stimulating hormone (α-MSH), also referred to as“melanocortin peptides.” Such peptides include the sequenceHis-Phe-Arg-Trp, His-D-Phe-Arg-Trp, or are homologs thereof, and can becyclic. A suitable melanocortin peptide isAc-Nle-cyclo-(-Asp-His-D-Phe-Arg-Trp-Lys)-OH. See U.S. Pat. No.6,051,555 to Hadley and International Patent Publication No. WO 01/00224to Blood et al., assigned to Palatin Technologies, Inc. Theaforementioned amino acid residues have their conventional meaning asgiven in Chapter 2422 of the Manual of Patent Examining Procedure(2000). Thus, “Arg” is arginine, “Nle” is norleucine, “His” ishistamine, “Phe” is phenylalanine, “D-Phe” is D-phenylalanine, “Trp” istryptophan, and “Ac” refers to an acetyl moiety, i.e., an acetyl moietypresent in a peptide or amino acid sequence that is acetylated.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is an endothelin antagonists, includingantagonists of any or all of the three isoforms of endothelin, i.e.,ET-1, ET-2, and ET-3, and are exemplified by: phenoxyphenylacetic acidsand derivatives thereof, such asN-(4-isopropylbenzene-sulfonyl)-α-(4-carboxy-2-n-propylphenoxy)-3,4-methylenedioxyphenylacetamide dipotassium salt,2-{(2,6-dipropyl-4-hydroxymethyl)-phenoxy}-2-(4-phenoxyphenyl)-aceticacid, 2-{(2,6-dipropyl-4-hydroxymethyl)phenoxy}-2-(4-phenylphenyl)aceticacid,2-{(2,6-dipropyl-4-hydroxymethyl)phenoxy}-2-(3-carboxyphenyl)-aceticacid,2-{(2,6-dipropyl-4-hydroxymethyl)phenoxy}-2-(3,4-ethylenedioxyphenyl)aceticacid,2-{(2,6-dipropyl-4-hydroxymethyl)phenoxy}-2-(3,4,5-trimethoxyphenyl)aceticacid,2-{(2,6-dipropyl-4-hydroxymethyl)phenoxy}-2-(3,4-methylenedioxyphenyl)aceticacid,N-(4-dimethylaminobenzenesulfonyl)-2-(4-methoxycarbonyl-2-propylphenoxy)-2-(3,4-methylenedioxyphenyl)acetamide,N-(2-methylbenzenesulfonyl)-2-(4-methoxycarbonyl-2-propylphenoxy)-2-(3,4-methylenedioxyphenyl)acetamide,N-(2-methoxycarbonyl-benzenesulfonyl)-2-(4-methoxy-carbonyl-2-propylphenoxy)-2-(3,4-methylenedioxy-phenyl)acetamide,N-(2-chlorobenzene-sulfonyl)-2-(4-methoxycarbonyl-2-propylphenoxy)-2-(3,4-methylenedioxyphenyl)acetamide,and others, as described in U.S. Pat. No. 5,565,485; and certainisooxazoles, oxazoles, thiazoles, isothiazoles and imidazoles, asdescribed, for example, in U.S. Pat. No. 6,136,828.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a peptidyl drug including the peptidylhormones activin, amylin, angiotensin, atrial natriuretic peptide (ANP),calcitonin, calcitonin gene-related peptide, calcitonin N-terminalflanking peptide, ciliary neurotrophic factor (CNTF), corticotropin(adrenocorticotropin hormone, ACTH), corticotropin-releasing factor (CRFor CRH), epidermal growth factor (EGF), follicle-stimulating hormone(FSH), gastrin, gastrin inhibitory peptide (GIP), gastrin-releasingpeptide, gonadotropin-releasing factor (GnRF or GNRH), growth hormonereleasing factor (GRF, GRH), human chorionic gonadotropin (hCH), inhibinA, inhibin B, insulin, luteinizing hormone (LH), luteinizinghormone-releasing hormone (LHRH), α-melanocyte-stimulating hormone,β-melanocyte-stimulating hormone, γ-melanocyte-stimulating hormone,melatonin, motilin, oxytocin (pitocin), pancreatic polypeptide,parathyroid hormone (PTH), placental lactogen, prolactin (PRL),prolactin-release inhibiting factor (PIF), prolactin-releasing factor(PRF), VIP, somatotropin (growth hormone, GH), somatostatin (SIF, growthhormone-release inhibiting factor, GIF), thyrotropin(thyroid-stimulating hormone, TSH), thyrotropin-releasing factor (TRH orTRF), thyroxine, and vasopressin. Other peptidyl drugs are thecytokines, e.g., colony stimulating factor 4, heparin bindingneurotrophic factor (HBNF), interferon-α, interferon α-2a, interferonα-2b, interferon α-n3, interferon-β, etc., interleukin-1, interleukin-2,interleukin-3, interleukin-4, interleukin-5, interleukin-6, etc., tumornecrosis factor, tumor necrosis factor-α, granuloycte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colony-stimulating factor, midkine (MD), andthymopoietin.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a selective androgen receptormodulators (SARMs) include LGD2226 and/or LGD1331, both available fromLigand Pharmaceuticals (San Diego, Calif.). See Negro-Villar et al. J.Clin. Endocrinol. & Metabol. 84(10):3459-62 (1999).

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a suitable neuropeptide includingbradykinin, kallidin, des-Arg9-bradykinin, des-Arg10-kallidin,des-Arg9-{Leu8}-bradykinin, {D-Phe7}-bradykinin, HOE 140, neuropeptideY, calcitonin gene-related peptide (cGRP), enkaphalins and relatedopioid peptides such as Met5-enkaphalin, Leu5-enkephalin, α-, β- andγ-endorphin, α- and β-neo-endorphin, and dynorphin, as well as theneurotransmitters GABA (γ-aminobutyric acid), glycine, glutamate,acetylcholine, dopamine, epinephrine, 5-hydroxytryptamine, substance P,serotonin, and catecholamines.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a suitable serotonin agonists include,but are not limited to 2-methyl serotonin, buspirone, ipsaperone,tiaspirone, gepirone, ergot alkaloids,8-hydroxy-(2-N,N-dipropyl-amino)-tetraline,1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane, cisapride, sumatriptan,m-chlorophenylpiperazine, trazodone, zacopride, mezacopride, andcombinations thereof. Suitable serotonin antagonists include, forexample, ondansetron, granisetron, metoclopramide, tropisetron,dolasetron, palonosetron, trimethobenzamide, methysergide, risperidone,ketanserin, ritanserin, clozapine, amitriptyline, MDL 100,907(R(+)-α-(2,3-dimethoxyphenyl)-1-{2-(4-fluorophenyl)ethyl}-4-piperidine-methanol)(Marion Merrell Dow), azatadine, cyproheptadine, fenclonine,chlorpromazine, mianserin and combinations thereof.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is an ergot alkaloids include ergotamineand ergotamine analogs, e.g., acetergamine, brazergoline, bromerguride,cianergoline, delorgotrile, dihydroergotamine, disulergine, ergonovine,ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile,lysergide, mesulergine, metergoline, metergotamine, nicergoline,pergolide, propisergide, proterguride and terguride.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a calcium channel blockers that aresuitable for use according to the present invention include, withoutlimitation, amlodipine, felodipine, isradipine, nicardipine, nifedipine,nimodipine, nisoldipine, nitrendipine, bepridil, diltiazem, verapamil,and combinations thereof. In some embodiments, the pharmaceuticalcompositions of the invention comprise an active agent that is apotassium channel openers include, but are not limited to, pinacidil,diazoxide, cromakalim, nicorandil, minoxidil,(N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridyl-guanidine (P-1075), andN-cyano-N′-(2-nitroxyethyl)-3-pridinecarboximidamidemonomethanesulfonate (KRN 2391).

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a potassium channel blockers includetedisamil, agitoxin-2, apamin, BDS-I, BDS-II, charybdotoxin,α-dendrotoxin, β-dendrotoxin, γ-dendrotoxin, δ-dendrotoxin,dendrotoxin-I, dendrotoxin-K, E-4031, iberiotoxin, kaliotoxin,MCD-peptide, margatoxin, noxiustoxin, paxilline, penitrem A,stichodactyla, tertiapin, tityustoxin K alpha, verruculogen, andcombinations thereof. Although all of the active agents are availablecommercially, most of the listed potassium channel blockers areavailable from Alomone Labs (Jerusalem, Israel).

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a dopamine agonist including, forexample, levodopa, bromocriptine, pergolide, apomorphine, piribedil,pramipexole, ropinirole, and combinations thereof. Dopamine antagonistsinclude, without limitation, spiroperidol, benperidol, trifluperidol,pimozide, fluphenazine, droperidol, haloperidol, thiothixene,trifluperazine, moperone, prochlorperazine, molindone, thioridazine,clozapine, chlorpromazine, promazine, sulpiride, clebopride,chlorpromazine, spiperone, flupenthixol, and combinations thereof.

In some embodiments, the pharmaceutical compositions of the inventioncomprise an active agent that is a non-androgenic steroid includingprogestins and estrogens. Suitable estrogens include synthetic andnatural estrogens such as: estradiol (i.e.,1,3,5-estratriene-3,17β-diol, or “17β-estradiol”) and its esters,including estradiol benzoate, valerate, cypionate, heptanoate,decanoate, acetate and diacetate; 17α-estradiol; ethinylestradiol (i.e.,17α-ethinylestradiol) and esters and ethers thereof, includingethinylestradiol 3-acetate and ethinylestradiol 3-benzoate; estriol andestriol succinate; polyestrol phosphate; estrone and its esters andderivatives, including estrone acetate, estrone sulfate, and piperazineestrone sulfate; quinestrol; mestranol; and conjugated equine estrogens.Suitable progestins include acetoxypregnenolone, allylestrenol,anagestone acetate, chlormadinone acetate, cyproterone, cyproteroneacetate, desogestrel, dihydrogesterone, dimethisterone, ethisterone(17α-ethinyltestosterone), ethynodiol diacetate, flurogestone acetate,gestadene, hydroxyprogesterone, hydroxyprogesterone acetate,hydroxyprogesterone caproate, hydroxymethylprogesterone,hydroxymethylprogesterone acetate, 3-ketodesogestrel, levonorgestrel,lynestrenol, medrogestone, medroxyprogesterone acetate, megestrol,megestrol acetate, melengestrol acetate, norethindrone, norethindroneacetate, norethisterone, norethisterone acetate, norethynodrel,norgestimate, norgestrel, norgestrienone, normethisterone, andprogesterone. It is generally desirable to co-administer a progestinalong with an estrogen so that the estrogen is not “unopposed.” As iswell known in the art, estrogen-based therapies are known to increasethe risk of endometrial hyperplasia and cancer, as well as the risk ofbreast cancer, in treated individuals. Co-administration of estrogenicagents with a progestin has been found to decrease the aforementionedrisks.

The pharmaceutical compositions of the present invention may alsoinclude one or more chemotherapeutic agents. Suitable chemotherapeuticagents include, but are not limited to, platinum coordination compounds,topoisomerase inhibitors, antibiotics, antimitotic alkaloids anddifluoronucleosides.

In one embodiment of the present invention, the chemotherapeutic agentis a platinum coordination compound. The term “platinum coordinationcompound” refers to any tumor cell growth inhibiting platinumcoordination compound that provides the platinum in the form of an ion.Suitable platinum coordination compounds include, but are not limitedto, cis-diamminediaquoplatinum (II)-ion;chloro(diethylenetriamine)-platinum (II) chloride;dichloro(ethylenediamine)-platinum (II); diammine(1,1-cyclobutanedicarboxylato) platinum (II) (carboplatin); spiroplatin;iproplatin; diammine (2-ethylmalonato)-platinum (II);ethylenediaminemalonatoplatinum (II); aqua(1,2-diaminodyclohexane)-sulfatoplatinum (II); (1,2-diaminocyclohexane)malonatoplatinum (II); (4-caroxyphthalato) (1,2-diaminocyclohexane)platinum (II); (1,2-diaminocyclohexane)-(isocitrato) platinum (II);(1,2-diaminocyclohexane) cis (pyruvato) platinum (II);(1,2-diaminocyclohexane) oxalatoplatinum (II); ormaplatin; andtetraplatin

In some embodiments, the VIP analog and the additional active agent oragents may be incorporated into a single formulation, or they may beadministered separately, either simultaneously or sequentially. In oneembodiment, an androgenic agent is administered prior to administrationof VIP or a VIP agonist, i.e., the androgenic agent is administered as apretreatment. In some embodiments, such a method involves administrationof an androgenic agent, e.g., via oral or topical (vulvar and/orvaginal) administration, followed by topical (again, vulvar and/orvaginal) administration of VIP or a VIP agonist.

In some embodiments, the formulations herein are administered by topicalapplication to the vulvar region and/or by vaginal drug administration.These pharmaceutical formulations may typically contain one or morepharmaceutically acceptable carriers suited to the particular type offormulation, i.e., gel, ointment, suppository, or the like. The vehiclesare comprised of materials of naturally occurring or synthetic originthat do not adversely affect the active agent or other components of theformulation. Suitable carriers for use herein include water, silicone,waxes, petroleum jelly, polyethylene glycol, propylene glycol,liposomes, sugars such as mannitol and lactose, and a variety of othermaterials, again depending, on the specific type of formulation used. Asdescribed in Section IV, infra, dosage forms used for administration tothe vulvar region and/or vagina may be used to deliver drug on anas-needed, on-demand basis, and/or throughout an extended, sustainedrelease profile.

The pharmaceutical compositions may also include a chemical compound toenhance permeation of the active agent through the mucosal tissue, i.e.,a “permeation enhancer.” Suitable permeation enhancers include thosegenerally useful in conjunction with topical, transdermal ortransmucosal drug delivery. Examples of suitable permeation enhancersinclude the following: sulfoxides such as dimethylsulfoxide (DMSO) anddecylmethylsulfoxide (C10MSO); ethers such as diethylene glycolmonoethyl ether (available commercially as TRANSCUTOL® (Gattefosse S.A., Saint-Priest, France) and diethylene glycol monomethyl ether;surfactants such as sodium laurate, sodium lauryl sulfate,cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231,182, 184), TWEEN® (20, 40, 60, 80) (ICI Chemicals, Bridgewater, N.J.),and lecithin (U.S. Pat. No. 4,783,450); the 1-substitutedazacycloheptan-2-ones, particularly 1-n-dodecylcyclazα-cycloheptan-2-one(available under the trademark AZONE® (Durham Pharmaceuticals, LLC,Durham, N.C.); see U.S. Pat. Nos. 3,989,816, 4,316,893, 4,405,616 and4,557,934); alcohols such as ethanol, propanol, octanol, decanol, benzylalcohol, and the like; fatty acids such as lauric acid, oleic acid andvaleric acid; fatty acid esters such as isopropyl myristate, isopropylpalmitate, methylpropionate, and ethyl oleate; polyols and estersthereof such as propylene glycol, ethylene glycol, glycerol, butanediol,polyethylene glycol, and polyethylene glycol monolaurate (PEGML; see,e.g., U.S. Pat. No. 4,568,343); amides and other nitrogenous compoundssuch as urea, dimethylacetamide (DMA), dimethylformamide (DMF),2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine; terpenes; alkanones; and organic acids, particularlysalicylic acid and salicylates, citric acid and succinic acid. Mixturesof two or more enhancers may also be used.

In some embodiments, the pharmaceutical compositions may include anenzyme inhibitor, i.e., a compound effective to inhibit enzymes presentin the vagina or vulvar area that could degrade or metabolize the activeagent. That is, inhibitors of enzymes that decrease or eliminate theactivity of the active agent may be included in the formulation so as toeffectively inhibit the action of those enzymes. Such compounds include,for example, fatty acids, fatty acid esters, and NAD inhibitors.

In some embodiments, the pharmaceutical composition may be in the formof an ointment, cream, emulsion, lotion, gel, solid, solution,suspension, foam or liposomal formulation. Alternatively, theformulations may be contained within avaginal ring (e.g., as disclosedin U.S. Pat. No. 5,188,835 to Lindskog et al., assigned to KabiPharmacia AB), or within a tampon, suppository, sponge, pillow, puff, orosmotic pump system; these platforms are useful solely for vaginaldelivery. Ointments are semisolid preparations that are typically basedon petrolatum or other petroleum derivatives. The specific ointment baseto be used, as will be appreciated by those skilled in the art, is onethat will provide for optimum drug delivery. As with other carriers orvehicles, an ointment base should be inert, stable, non irritating andnonsensitizing. As explained in Remington: The Science and Practice ofPharmacy, supra, at pages 1034-1038, ointment bases may be grouped infour classes: oleaginous bases; emulsifiable bases; emulsion bases; andwater-soluble bases. Oleaginous ointment bases include, for example,vegetable oils, fats obtained from animals, and semisolid hydrocarbonsobtained from petroleum. Emulsifiable ointment bases, also known asabsorbent ointment bases, contain little or no water and include, forexample, hydroxystearin sulfate, anhydrous lanolin and hydrophilicpetrolatum. Emulsion ointment bases are either water-in-oil (W/O)emulsions or oil-in-water (O/W) emulsions, and include, for example,cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Suitablewater-soluble ointment bases are prepared from polyethylene glycols ofvarying molecular weight; again, reference may be had to Remington: TheScience and Practice of Pharmacy for further information.

In one aspect of the invention, a method is provided for treating orpreventing cancer comprising administering any of the disclosedcompositions disclosed herein in a therapeutically or prophylacticallyeffective amount. In some embodiments, a method is provided for treatingor preventing one or a combination of the following cancer types: smalllung cell cancer, exocrine pancreatic tumors, colorectal carcinoma,gastric carcinoma, hepatocellular carcinoma, esophageal carcinoma, renalcell carcinoma, prostate carcinoma, urinary bladder carcinoma, livercarcinoma, ductal pancreatic cancer, breast carcinoma, ovariancarcinoma, non-hodgkin's lymphoma, meningioma, GEP tumors(differentiated and undifferentiated), pituitary adenoma, endometrialcancer, astrocytoma, giloblastoma, non-small cell lung cancer,pancreatic cancer, melanoma, renal cancer, neuroblastoma, leukima,prostate cancer

In one aspect of the invention, a method is provided for treating sexualdysfunction in a female individual comprising administering to thevagina and/or vulvar area a pharmaceutical formulation comprising a VIPfamily analog. In some embodiments, the VIP family analog is avasodilator, with vasodilators selected from the group consisting of VIPand vasoactive intestinal polypeptide analogs and combinations of any ofthe foregoing. Any number of drug delivery platforms may be used, e.g.,suppositories, ointments, creams, gels, solutions and the like. Also,one or more additional types of drugs, i.e., pharmacologically activeagents may be incorporated into the pharmaceutical formulations. Inother aspects of the invention, vaginal administration of a vasoactiveagent as just described is used to improve vaginal muscle tone andtissue health, to enhance vaginal lubrication, or to minimize collagenmisdeposition resulting from hypoxia as well as the associated lack ofelasticity resulting from the collagen misdeposition.

In another embodiment of the invention, a method is provided forimproving memory by administering a VIP family analog.

In another aspect of the invention, pharmaceutical compositions anddosage forms are provided for carrying out the aforementioned methods.The compositions and dosage forms contain a vasoactive agent asdescribed above, a pharmaceutically acceptable vehicle, and, optionally,one or more additional pharmacologically active agents. The formulationscontain a therapeutically effective amount of the active agent, or atherapeutically effective concentration of the active agent, i.e., aconcentration that provides a therapeutically effective amount of activeagent upon administration of a selected volume of composition.

The subject can be any animal, including but not necessarily limited tomammals such as a human, mouse, rat, hamster, guinea pig, rabbit, cat,dog, monkey, cow, horse, pig, and the like. In some embodiments, thesubject is a human.

According to some embodiments of the invention, the formulation may besupplied as part of a kit. The kit comprise comprising an analog,wherein the analog comprises an α-amino acid and at least one β-aminoacid. In another embodiment, the kit comprises a pharmaceuticallyacceptable salt of an analog with a rehydration mixture. In anotherembodiment, the pharmaceutically acceptable salt of an analog are in onecontainer while the rehydration mixture is in a second container. Therehydration mixture may be supplied in dry form, to which water or otherliquid solvent may be added to form a suspension or solution prior toadministration. Rehydration mixtures are mixtures designed to solubilizea lyophilized, insoluble salt of the invention prior to administrationof the composition to a subject takes at least one dose of a purgative.In another embodiment, the kit comprises a pharmaceutically acceptablesalt in orally available pill form.

The kit may contain two or more containers, packs, or dispenserstogether with instructions for preparation and administration. In someembodiments, the kit comprises at least one container comprising thepharmaceutical composition or compositions described herein and a secondcontainer comprising a means for delivery of the compositions such as asyringe. In some embodiments, the kit comprises a composition comprisingan analog in solution or lyophilized or dried and accompanied by arehydration mixture. In some embodiments, the analog and rehydrationmixture may be in one or more additional containers.

The compositions included in the kit may be supplied in containers ofany sort such that the shelf-life of the different components arepreserved, and are not adsorbed or altered by the materials of thecontainer. For example, suitable containers include simple bottles thatmay be fabricated from glass, organic polymers, such as polycarbonate,polystyrene, polypropylene, polyethylene, ceramic, metal or any othermaterial typically employed to hold reagents or food; envelopes, thatmay consist of foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, and syringes. Thecontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components of thecompositions to mix. Removable membranes may be glass, plastic, rubber,or other inert material.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrates, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zipdisc, videotape, audio tape, or other readable memory storage device.Detailed instructions may not be physically associated with the kit;instead, a user may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

In another embodiment, a packaged kit is provided that contains thepharmaceutical formulation to be administered, i.e., a pharmaceuticalformulation containing VIP analog or a for enhancing female sexualdesire and responsiveness, a container (e.g., a vial, a bottle, a pouch,an envelope, a can, a tube, an atomizer, an aerosol can, etc.),optionally sealed, for housing the formulation during storage and priorto use, and instructions for carrying out drug administration in amanner effective to enhance sexual desire and responsiveness. Theinstructions will typically be written instructions on a package insert,a label, and/or on other components of the kit.

Depending on the type of formulation and the intended mode ofadministration, the kit may also include a device for administering theformulation (e.g., a transdermal delivery device). The administrationdevice may be a dropper, a swab, a stick, or the nozzle or outlet of anatomizer or aerosol can. The formulation may be any suitable formulationas described herein. For example, the formulation may be an oral dosageform containing a unit dosage of the active agent, or a gel or ointmentcontained within a tube. The kit may contain multiple formulations ofdifferent dosages of the same agent. The kit may also contain multipleformulations of different active agents.

The present kits will also typically include means for packaging theindividual kit components, i.e., the pharmaceutical dosage forms, theadministration device (if included), and the written instructions foruse. Such packaging means may take the form of a cardboard or paper box,a plastic or foil pouch, etc.

The invention relates to the use of an analog in the preparation of amedicament for treating or preventing chronic obstructive pulmonarydisease, pulmonary hypertension, primary arterial hypertension,pulmonary hypertension associated to post-ventricular septal defect,idiopathic pulmonary fibrosis, idiopathic pulmonary arterialhypertension, CREST syndrome—Calcinosis; Raynaud's disease; loss ofmuscle control of the Esophagus; Sclerodactyly; Telangiectasia, Acuterespiratory distress, congestive heart failure, chronic obstructedpulmonary disorder, asthma, chronic obstructive pulmonary disease,sarcoidosis, small cell lung carcinoma, autoimmune disease, inflammatorydisease, sepsis, Hirschsprung's Disease, sexual dysfunction, erectiledysfunction, Parkinson's disease, Alzheimer's disease, circadian rhythmdysfunction, pain, colorectal cancer, hepatocellular cancer, elevatedblood pressure levels, elevated blood glucose levels, hyperglycemia,diabetes, insulin resistance, metabolic acidosis, obesity, Type Idiabetes, Type II diabetes Multiple Sclerosis, osteoporosis, Sjogren'ssyndrome, pancreatitis, uveoretinitis, osteoporosis, female sexualdysfunction due to administration of a medication that causes onset ofor exacerbates symptoms of pulmonary hypertension, primary arterialhypertension, pulmonary hypertension associated to post-ventricularseptal defect, idiopathic pulmonary fibrosis, idiopathic pulmonaryarterial hypertension, CREST syndrome—Calcinosis; Raynaud's disease;loss of muscle control of the Esophagus; Sclerodactyly; Telangiectasia,Acute respiratory distress, congestive heart failure, chronic obstructedpulmonary disorder, asthma, chronic obstructive pulmonary disease,sarcoidosis, small cell lung carcinoma, autoimmune disease, inflammatorydisease, sepsis, Hirschsprung's Disease, sexual dysfunction, erectiledysfunction, Parkinson's disease, Alzheimer's disease, circadian rhythmdysfunction, pain, colorectal cancer, hepatocellular cancer, elevatedblood pressure levels, elevated blood glucose levels, hyperglycemia,diabetes, insulin resistance, metabolic acidosis, obesity, Type Idiabetes, Type II diabetes Multiple Sclerosis, osteoporosis, Sjogren'ssyndrome, pancreatitis, uveoretinitis, osteoporosis, female sexualdysfunction in a subject in need thereof. In some embodiments, theinvention relates to compositions comprising a VIP family analog fortreatment or prevention of chronic obstructive pulmonary disease,pulmonary hypertension, primary arterial hypertension, pulmonaryhypertension associated to post-ventricular septal defect, idiopathicpulmonary fibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small cell lungcarcinoma, autoimmune disease, inflammatory disease, sepsis,Hirschsprung's Disease, sexual dysfunction, erectile dysfunction,Parkinson's disease, Alzheimer's disease, circadian rhythm dysfunction,pain, colorectal cancer, hepatocellular cancer, elevated blood pressurelevels, elevated blood glucose levels, hyperglycemia, diabetes, insulinresistance, metabolic acidosis, obesity, Type I diabetes, Type IIdiabetes Multiple Sclerosis, osteoporosis, Sjogren's syndrome,pancreatitis, uveoretinitis, osteoporosis, female sexual dysfunction ina subject in need thereof.

The present invention relates to inhibiting secretion of TNF-α in asubject comprising administering a composition comprising an analog to asubject, wherein said analog comprises an α-amino acid and at least oneβ-amino acid. In some embodiments the analog is a VIP family analog. Insome embodiments the analog is a VIP analog.

The present invention relates to inhibiting binding of VIP to a VIPreceptor in a subject comprising administering a composition comprisingan analog to a subject, wherein said analog comprises an α-amino acidand at least one β-amino acid. In some embodiments the analog is a VIPfamily analog. In some embodiments the analog is a VIP analog.The present invention relates to inhibiting biological effect of GHRH ina subject comprising administering a composition comprising an analog toa subject, wherein said analog comprises an α-amino acid and at leastone β-amino acid. In some embodiments the analog is a VIP family analog.In some embodiments the analog is a VIP analog.The present invention relates to inhibiting chemotaxis of T cells in asubject comprising administering a composition comprising an analog to asubject, wherein said analog comprises an α-amino acid and at least oneβ-amino acid. In some embodiments the analog is a VIP family analog. Insome embodiments the analog is a VIP analog.The present invention relates to inhibiting expression of LPS in asubject comprising administering a composition comprising an analog to asubject, wherein said analog comprises an α-amino acid and at least oneβ-amino acid. In some embodiments the analog is a VIP family analog. Insome embodiments the analog is a VIP analog.The present invention relates to modulating the amount of cyclic cAMP ina subject comprising administering a composition comprising an analog toa subject, wherein said analog comprises an α-amino acid and at leastone β-amino acid. In some embodiments the analog is a VIP family analog.In some embodiments the analog is a VIP analog.The present invention relates to increasing the activity or expressionof adenylate cyclase in a subject comprising administering a compositioncomprising an analog to a subject, wherein said analog comprises anα-amino acid and at least one β-amino acid. In some embodiments theanalog is a VIP family analog. In some embodiments the analog is a VIPfamily analog and a VPAC1 antagonist. In some embodiments the analog isa VIP family analog. and a VPAC2 agonist. In some embodiments the analogis a VIP analog. In some embodiments, the composition or pharmaceuticalcomposition of the claimed invention comprises a VIP analog, wherein theVIP analog is a VIPR1 agonist, and has substantially reduced selectivityor no selectivity for VIPR2 or PAC1 receptors. In some embodiments, thecomposition or pharmaceutical composition of the claimed inventioncomprises a VIP analog, wherein the VIP analog is a PAC1 agonist, andhas substantially reduced selectivity or no selectivity for VIPR2 orVIPR1 receptors. In some embodiments, the composition or pharmaceuticalcomposition of the claimed invention comprises a VIP analog, wherein theVIP analog is a VIPR2 agonist, and has substantially reduced selectivityor no selectivity for VIPR1 or PAC1 receptors. In some embodiments, thecomposition or pharmaceutical composition of the claimed inventioncomprises a VIP analog, wherein the VIP analog is a VIPR2 antagonist,but does not antagonize VIPR1 or PAC1 receptors. In some embodiments,the composition or pharmaceutical composition of the claimed inventioncomprises a VIP analog, wherein the VIP analog is a VIPR1 antagonist,but does not antagonize VIPR2 or PAC1 receptors. In some embodiments,the composition or pharmaceutical composition of the claimed inventioncomprises a VIP analog, wherein the VIP analog is a PAC1 antagonist, butdoes not antagonize VIPR2 or VIPR1 receptors. Any of the above-mentionedselective agonist or antagonists may be used in any of the method claimsprovided herein.

The present invention relates to modulating the amount of PLD in thenervous system of a subject comprising administering a compositioncomprising an analog to a subject, wherein said analog comprises anα-amino acid and at least one β-amino acid. In some embodiments theanalog is a VIP family analog. In some embodiments the analog is a VIPanalog.

The present invention relates to modulating the amount of antibodyproduction of a B cell in a subject comprising administering acomposition comprising an analog to a subject, wherein said analogcomprises an α-amino acid and at least one β-amino acid. In someembodiments the analog is a VIP family analog. In some embodiments theanalog is a VIP analog.

The present invention relates to modulating the amount of antibodyproduction of a B cell or a B cell hybridoma cell in vitro comprisingtreating a culture containing B cells or a hyvridoma with a compositioncomprising an analog to a subject, wherein said analog comprises anα-amino acid and at least one β-amino acid. In some embodiments theanalog is a VIP family analog. In some embodiments the analog is a VIPanalog.

The present invention relates to modulating the immune response of asubject comprising administering a subject with a composition comprisingan analog to a subject, wherein said analog comprises an α-amino acidand at least one β-amino acid. In some embodiments the analog is a VIPfamily analog. In some embodiments the analog is a VIP analog.

The present invention relates to modulating the activation of cysticfibrosis transmembrane conductance regulator (CFTR) in a subjectcomprising administering a subject with a composition comprising ananalog to a subject, wherein said analog comprises an α-amino acid andat least one β-amino acid. In some embodiments the analog is a VIPfamily analog. In some embodiments the analog is a VIP analog.

The present invention also relates measuring the modulation of activityof a VIP receptor molecule by measuring receptor activity comprising:

a) contacting a human VIP family receptor with a VIP family analog,wherein the analog comprises an α-amino acid and at least one β-aminoacid;

b) measuring the association of the VIP family analog to the VIPreceptor in the presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP family analog to thehuman VIP receptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound.

The present invention also relates identifying a modulator of activityof a VIP receptor molecule by measuring receptor activity comprising:

a) contacting a human VIP family receptor with a VIP family analog,wherein said analog comprises an α-amino acid and at least one β-aminoacid;

b) measuring the association of the VIP family analog to the VIPreceptor in the presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP family analog to thehuman VIP receptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound.

The present invention also relates to a method of measuring themodulation of activity of a human VIP receptor molecule by measuringreceptor activity comprising:

a) contacting a human VIP family receptor with a VIP analog, wherein theanalog comprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the VIP receptor inthe presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the human VIPreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound.

The present invention also relates identifying a modulator of activityof a VIP family receptor molecule by measuring receptor activitycomprising:

a) contacting a human VIP family receptor with a VIP analog, whereinsaid analog comprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the VIP receptor inthe presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the human VIPreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound. In some embodiments, the VIP family receptor ischosen from VIPR1, VIPR2, VPAC₁, VPAC₂ or PAC₁.

The present invention also relates identifying a modulator of activityof a VIP family receptor molecule by measuring receptor activitycomprising:

a) contacting a VIP family receptor with a VIP analog in a knownconcentration, wherein said analog comprises an α-amino acid and atleast one β-amino acid;

b) measuring the binding affinity of the VIP analog to the VIP familyreceptor in the presence and absence of a compound that binds to the VIPfamily receptor; and

c) comparing the binding affinity of the VIP analog to the VIP receptorin the presence of a compound that binds to the VIP family receptor tothe binding affinity of the VIP analog to the VIP receptor in theabsence of a compound that binds to the VIP family receptor. In someembodiments, the VIP family receptor is chosen from VIPR1, VIPR2, VPAC₁,VPAC₂ or PAC₁.

The invention also relates to the use of an analog with selectivity forVPAC1, PAC1, or VPAC2 in the preparation of a medicament for treating orpreventing chronic obstructive pulmonary disease, pulmonaryhypertension, primary arterial hypertension, pulmonary hypertensionassociated to post-ventricular septal defect, idiopathic pulmonaryfibrosis, idiopathic pulmonary arterial hypertension, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small cell lungcarcinoma, autoimmune disease, inflammatory disease, sepsis,Hirschsprung's Disease, sexual dysfunction, erectile dysfunction,Parkinson's disease, Alzheimer's disease, circadian rhythm dysfunction,pain, colorectal cancer, hepatocellular cancer, elevated blood glucoselevels, elevated blood pressure, hyperglycemia, diabetes, insulinresistance, metabolic acidosis, obesity, Type I diabetes, Type IIdiabetes Multiple Sclerosis, osteoporosis, Sjogren's syndrome,pancreatitis, uveoretinitis, osteoporosis, female sexual dysfunction dueto administration of a medication that causes onset of or exacerbatessymptoms of pulmonary hypertension, primary arterial hypertension,pulmonary hypertension associated to post-ventricular septal defect,idiopathic pulmonary fibrosis, idiopathic pulmonary arterialhypertension, CREST syndrome—Calcinosis; Raynaud's disease; loss ofmuscle control of the Esophagus; Sclerodactyly; Telangiectasia, Acuterespiratory distress, congestive heart failure, chronic obstructedpulmonary disorder, asthma, chronic obstructive pulmonary disease,sarcoidosis, small cell lung carcinoma, autoimmune disease, inflammatorydisease, sepsis, Hirschsprung's Disease, sexual dysfunction, erectiledysfunction, Parkinson's disease, Alzheimer's disease, circadian rhythmdysfunction, pain, colorectal cancer, hepatocellular cancer, elevatedblood pressure levels, elevated blood glucose levels, hyperglycemia,diabetes, insulin resistance, metabolic acidosis, obesity, Type Idiabetes, Type II diabetes Multiple Sclerosis, osteoporosis, Sjogren'ssyndrome, pancreatitis, uveoretinitis, osteoporosis, female sexualdysfunction in a subject in need thereof. In some embodiments, theinvention relates to compositions comprising a VIP family analog withselectivity for VPAC1, PAC1, or VPAC2 for treatment or prevention ofchronic obstructive pulmonary disease, pulmonary hypertension, primaryarterial hypertension, pulmonary hypertension associated topost-ventricular septal defect, idiopathic pulmonary fibrosis,idiopathic pulmonary arterial hypertension, CREST syndrome—Calcinosis;Raynaud's disease; loss of muscle control of the Esophagus;Sclerodactyly; Telangiectasia, Acute respiratory distress, congestiveheart failure, chronic obstructed pulmonary disorder, asthma, chronicobstructive pulmonary disease, sarcoidosis, small cell lung carcinoma,autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels,hyperglycemia, diabetes, insulin resistance, metabolic acidosis,obesity, Type I diabetes, Type II diabetes Multiple Sclerosis,osteoporosis, Sjogren's syndrome, pancreatitis, uveoretinitis,osteoporosis, female sexual dysfunction in a subject in need thereof.

The present invention also relates to a method of treating or preventingcancer in a subject in need thereof comprising administering a VIPanalog to the subject, wherein said analog comprises an α-amino acid andat least one β-amino acid and wherein said analog is a VPAC1, VPAC2, orPAC1 receptor antagonist or agonist with increased selectivity for theVPAC1, VPAC2, or PAC1 receptor as compared to the other receptors. Insome embodiments, the cancer is chosen from the following: non-smallcell lung carcinoma, small cell lung carcinoma, colorectal carcinoma,breast carcinoma, gastric carcinoma, prostate carcinoma, livercarcinoma, ductal pancreatic carcinoma, bladder carcinoma, Non-Hodgkin'slymphoma, maningioma, leiomyoma, endometrial carcinoma,pheochromocytoma, paraganglioma. The present invention also relates to amethod of treating or preventing inflammatory disease comprisingadministering a VIP analog to a subject in need thereof, wherein saidanalog comprises an α-amino acid and at least one β-amino acid andwherein said analog is a VPAC1, VPAC2, or PAC1 receptor antagonist oragonist with increased selectivity for the VPAC1, VPAC2, or PAC1receptor as compared to the other receptors. In some embodiments theinflammatory disease is rheumatoid arthritis. In some embodiments, theVIP analog is administered at a therapeutically effective dose.

The present invention also relates to a method of treating or preventingcancer in a subject in need thereof comprising administering a VIPanalog to the subject, wherein said analog comprises an α-amino acid andat least one β-amino acid and wherein said analog is a VPAC1 receptorantagonist with increased selectivity for the VPAC1 receptor. Thepresent invention also relates to a method of treating or preventinginflammatory disease comprising administering a VIP analog to a subjectin need thereof, wherein said analog comprises an α-amino acid and atleast one β-amino acid and wherein said analog is a VPAC1 receptorantagonist with increased selectivity for the VPAC1 receptor. In someembodiments the inflammatory disease is rheumatoid arthritis. In someembodiments, the VIP analog is administered at a therapeuticallyeffective dose.

The present invention also relates to a method of treating or preventingsmall cell lung carcinoma comprising administering a VIP analog to asubject in need thereof, wherein said analog comprises an α-amino acidand at least one β-amino acid and wherein said analog is a VPAC1, VPAC2,or PAC1 receptor antagonist or agonist with increased selectivity for atleast one VPAC1, VPAC2, or PAC1 receptor. The present invention alsorelates to a method of treating or preventing inflammatory diseasecomprising administering a VIP analog to a subject in need thereof,wherein said analog comprises an α-amino acid and at least one β-aminoacid and wherein said analog is a VPAC1, VPAC2, or PAC1 receptorantagonist or agonist with increased selectivity for at least one of thefollowing: VPAC1, VPAC2, or PAC1 receptors. In some embodiments, the VIPanalog is administered at a therapeutically effective dose.

The present invention also relates to a method of treating or preventingprimary arterial hypertension (PAH) comprising administering a VIPanalog to a subject in need thereof, wherein said analog comprises anα-amino acid and at least one β-amino acid and wherein said analog is aVPAC1, VPAC2, or PAC1 receptor antagonist or agonist with increasedselectivity for at least one VPAC1, VPAC2, or PAC1 receptor. The presentinvention relates to a method of treating or preventing inflammatorydisease comprising administering a VIP analog to a subject in needthereof, wherein said analog comprises an α-amino acid and at least oneβ-amino acid and wherein said analog is a VPAC1, VPAC2, or PAC1 receptorantagonist or agonist with increased selectivity for at least one of thefollowing: VPAC1, VPAC2, or PAC1 receptors as compared to itsselectivity for the other receptors. In some embodiments, the VIP analogis administered at a therapeutically effective dose.

The present invention also relates to a method of treating or preventinginflammatory disease comprising administering a VIP analog to a subjectin need thereof, wherein said analog comprises an α-amino acid and atleast one β-amino acid and wherein said analog is a VPAC1 receptoragonist with increased selectivity for the VPAC1 receptor. The presentinvention relates to a method of treating or preventing inflammatorydisease comprising administering a VIP analog to a subject in needthereof, wherein said analog comprises an α-amino acid and at least oneβ-amino acid and wherein said analog is a VPAC1 receptor agonist withincreased selectivity for the VPAC1 receptor. In some embodiments theinflammatory disease is rheumatoid arthritis. In some embodiments, theVIP analog is administered at a therapeutically effective dose.

The present invention also relates to a method of treating or preventinginflammatory disease comprising administering a VIP analog to a subjectin need thereof, wherein said analog comprises an α-amino acid and atleast one β-amino acid and wherein said analog is a VPAC2 receptoragonist with increased selectivity for the VPAC2 receptor. The presentinvention relates to a method of treating or preventing inflammatorydisease comprising administering a VIP analog to a subject in needthereof, wherein said analog comprises an α-amino acid and at least oneβ-amino acid and wherein said analog is a VPAC2 receptor agonist withincreased selectivity for the VPAC2 receptor. In some embodiments theinflammatory disease is rheumatoid arthritis. In some embodiments, theVIP analog is administered at a therapeutically effective dose.

The present invention also relates to a method of treating or preventingchronic obstructive pulmonary disease, pulmonary hypertension, primaryarterial hypertension, pulmonary hypertension associated topost-ventricular septal defect, idiopathic pulmonary fibrosis,idiopathic pulmonary arterial hypertension comprising administering aVIP analog with selectivity for VPAC2 to a subject in need thereof,wherein said analog comprises an α-amino acid and at least one β-aminoacid and wherein said analog is a VPAC2 receptor agonist with increasedselectivity to VPAC2 receptor. In all methods of treatment orprevention, analogs of the present invention may be administered intherapeutically effective doses.

The present invention relates to a method of treating or preventingchronic obstructive pulmonary disease (COPD) comprising administering aVIP analog to a subject in need thereof, wherein said analog comprisesan α-amino acid and at least one β-amino acid and wherein said analog isa VPAC1 receptor antagonist or agonist with increased selectivity forthe VPAC1 receptor. The present invention relates to a method oftreating or preventing COPD comprising administering a VIP analog to asubject in need thereof, wherein said analog comprises an α-amino acidand at least one β-amino acid and wherein said analog is a VPAC1receptor antagonist or agonist with increased selectivity for the VPAC1receptor. In some embodiments, the VIP analog is administered at atherapeutically effective dose via nebulizer or inhaler.

The invention also relates to a method of preventing or inhibitingactivation of alveolar macrophages comprising administering a VIP analogto a subject, wherein said analog comprises an α-amino acid and at leastone β-amino acid and wherein said analog is a VPAC1 receptor antagonistor agonist with increased selectivity for the VPAC1 receptor. In someembodiments, the VIP analog is administered at a therapeuticallyeffective dose via nebulizer or inhaler.

The present invention relates to a method of treating or preventingchronic obstructive pulmonary disease (COPD) comprising administering aVIP analog to a subject in need thereof, wherein said analog comprisesan α-amino acid and at least one β-amino acid and wherein said analog isa VPAC2 receptor agonist with increased selectivity for the VPAC2receptor. The present invention relates to a method of treating orpreventing COPD comprising administering a VIP analog to a subject inneed thereof, wherein said analog comprises an α-amino acid and at leastone β-amino acid and wherein said analog is a VPAC2 receptor agonistwith increased selectivity for the VPAC2 receptor. In some embodiments,the VIP analog is administered at a therapeutically effective dose vianebulizer or inhaler. The invention relates to a method of preventing orinhibiting activation of alveolar macrophages comprising administering aVIP analog to a subject, wherein said analog comprises an α-amino acidand at least one β-amino acid and wherein said analog is a VPAC2receptor agonist with increased selectivity for the VPAC2 receptor. Insome embodiments, the VIP analog is administered at a therapeuticallyeffective dose via nebulizer or inhaler.

The present invention also relates to methods of identifying a selectivemodulator of activity of a VIP family receptor molecule by measuringreceptor activity comprising:

a) contacting a human VIP family receptor with a VIP analog, whereinsaid analog comprises an α-amino acid and at least one β-amino acid;

b) measuring the association of the VIP analog to the VIP receptor inthe presence and absence of an unknown compound; and

c) comparing the rate of association of the VIP analog to the human VIPreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the human VIP receptor in the absenceof an unknown compound.

The present invention also relates to methods of identifying a selectivemodulator of activity of a VIP family receptor molecule by measuringreceptor activity comprising:

a) contacting a first and a second VIP family receptor with a VIP analogin a known concentration, wherein said analog comprises an α-amino acidand at least one β-amino acid;

b) measuring the rate association of the VIP analog to the first andsecond VIP receptors in the presence and absence of an unknown compound;and

c) comparing the rate of association of the VIP analog to the first VIPreceptor in the presence of an unknown compound to the rate ofassociation of the VIP analog to the second VIP receptor in the absenceof an unknown compound.

The present invention also relates to methods of identifying a selectivemodulator of activity of a VIP family receptor molecule by measuringreceptor activity comprising:

a) contacting a first and a second VIP family receptor with a VIP analogin a known concentration, wherein said analog comprises an α-amino acidand at least one β-amino acid;

b) measuring the binding affinity of the VIP analog to the first andsecond VIP receptors in the presence and absence of an unknown compound;and

c) comparing the binding affinity of the VIP analog to the first VIPreceptor in the presence of an unknown compound to the binding affinityof the VIP analog to the second VIP receptor in the absence of anunknown compound. In some embodiments, the VIP family receptor is chosenfrom VIPR1, VIPR2, VPAC₁, VPAC₂ or PAC₁.

The present invention also relates to methods of inhibiting the immuneresponse against a transplanted organ in a subject, wherein the subjectis an organ donor recipient. in some embodiments, the subject is amammal. In some embodiments, the subject is a human. In someembodiments, the subject is a human experiencing organ rejection aftertransplantation.

In another embodiment, the present invention also relates to a methodfor inhibiting the growth of a tumor cell, the method comprising:contacting the tumor cell with an effective amount of a VIP familyanalog, wherein the VIP family analog or functional fragment thereofcomprises at least one β-amino acid. In some embodiments, the methodcomprises contacting the tumor cell with an effective amount of acombination of a chemotherapeutic agent and a VIP family analog. In someembodiments, the VIP analog is a VIP analog. Suitable chemotherapeuticagents include, but are not limited to, platinum coordination compounds,topoisomerase inhibitors, antibiotics, antimitotic alkaloids anddifluoronucleosides. In some embodiments, the VIP analog is a VPAC1antagonist with selectivity for VPAC1. In some embodiments, the tumorcell is a tumor cell derived from a breast cancer, a lung cancer, acolon cancer, a prostate cancer, or a pancreatic cancer.

In another embodiment, the present invention also relates to a method ofinhibiting the growth of a tumor cell in a mammalian subject in needthereof, the method comprising: administering to the subject aneffective amount of a VIP family analog or functional fragment thereof,wherein the VIP family analog or functional fragment thereof comprisesat least one β-amino acid. In some embodiments, the method comprisesadministering to the subject an effective amount of a combination of achemotherapeutic agent and a VIP family analog. In some embodiments, theVIP analog is a VIP analog. In some embodiments, the tumor cell is atumor cell derived from a breast cancer, a lung cancer, a colon cancer,a prostate cancer, hepatic cancer (HCC) or a pancreatic cancer. Suitablechemotherapeutic agents include, but are not limited to, platinumcoordination compounds, topoisomerase inhibitors, antibiotics,antimitotic alkaloids and difluoronucleosides.

The present invention also relates to a method of treating or preventingcancer cell growth in a subject in need thereof comprising the steps of:administering a VIP analog or functional fragment thereof the subject,wherein the VIP analog or functional fragment comprises at least oneβ-amino acid, wherein the VIP analog or functional fragment thereof isselective or has increased selectivity to VPAC₁; wherein the VIP analogis a VPAC₁ antagonist; and wherein the cancer cell is a bladder, breast,colon, liver, lung, prostate, stomach, thyroid or uterine cancer cell.The present invention relates to a method of treating or preventingcancer in a subject in need thereof comprising the steps of:administering a VIP analog or functional fragment thereof the subject,wherein the VIP analog or functional fragment comprises at least oneβ-amino acid, wherein the VIP analog or functional fragment thereof isselective or has increased selectivity to VPAC₁; wherein the VIP analogis a VPAC₁ antagonist; and wherein the cancer is a bladder, breast,colon, liver, lung, prostate, stomach, thyroid, hepatocellular, oruterine cancer. In some embodiments, the cancer has been diagnosed asbeing malignant. In some embodiments, the subject may have an increasedrisk or increased susceptibility to contracting a malignant cancer.

The present invention also relates to a method of treating or preventingcancer cell growth in a subject in need thereof comprising the steps of:administering a VIP analog or functional fragment thereof the subject,wherein the VIP analog or functional fragment comprises at least oneβ-amino acid, wherein the VIP analog or functional fragment thereof isselective or has increased selectivity to VPAC₂; wherein the VIP analogis a VPAC₂ antagonist; and wherein the cancer cell is a lung, breast,stomach cancer cell. In some embodiments the cancer cell is derived froma stomach leiomyoma.

The present invention also relates to a method of treating or preventingcancer in a subject in need thereof comprising the steps of:administering a VIP analog or functional fragment thereof the subject,wherein the VIP analog or functional fragment comprises at least oneβ-amino acid, wherein the VIP analog or functional fragment thereof isselective or has increased selectivity to VPAC₂; wherein the VIP analogis a VPAC₂ antagonist; and wherein the cancer a lung, breast, stomach,or heptocellular cancer. In some embodiments, the cancer has beendiagnosed as being malignant. In some embodiments, the subject may havean increased risk or increased susceptibility to contracting a malignantcancer.

The present invention also relates to a method of treating or preventingairway constriction comprising administering a VIP analog or functionalfragment thereof to a subject in need thereof, wherein said analogcomprises an α-amino acid and at least one β-amino acid and wherein saidanalog is a VPAC2 receptor agonist. In some embodiments, the VIP analogor functional fragment thereof has increased selectivity to VPAC2receptor. In all methods of treatment or prevention, analogs of thepresent invention may be administered in therapeutically effectivedoses.

The present invention also relates to a method of treating or preventingasthma, comprising administering a VIP analog or functional fragmentthereof to a subject in need thereof, wherein said analog comprises anα-amino acid and at least one β-amino acid and wherein said analog is aVPAC2 receptor agonist. In some embodiments, the VIP analog orfunctional fragment thereof has increased selectivity to VPAC2 receptor.In all methods of treatment or prevention, analogs of the presentinvention may be administered in therapeutically effective doses. Insome embodiments, the VIP analog or functional fragment thereof may beadministered via an inhaler or nebulizer.

The present invention also relates to a method of treating or preventingcancer cell growth in a subject in need thereof comprising the steps of:administering a VIP analog or functional fragment thereof the subject,wherein the VIP analog or functional fragment comprises at least oneβ-amino acid, wherein the VIP analog or functional fragment thereof isselective or has increased selectivity to PAC₁; wherein the VIP analogis a PAC₁ antagonist; and wherein the cancer cell is a nerve cell,adrenal cell, pituitary cell, or breast cell. The present invention alsorelates to a method of treating or preventing cancer in a subject inneed thereof comprising the steps of: administering a VIP analog orfunctional fragment thereof the subject, wherein the VIP analog orfunctional fragment comprises at least one β-amino acid, wherein the VIPanalog or functional fragment thereof is selective or has increasedselectivity to PAC₁; wherein the VIP analog is a PAC₁ antagonist; andwherein the cancer is a glioblastoma, neuroblastoma, adrenal, pituitary,catecholamine-VIPg tumors, pheochromocytomas, paragangliomas,endometrial cancers, or breast cancer. In some embodiments, the cancerhas been diagnosed as being malignant. In some embodiments, the subjectmay have an increased risk or increased susceptibility to contracting amalignant cancer.

The invention also relates to methods of treating or preventing theaforementioned diseases using the analogs of the present invention. Anyanalog described in the present invention may or may not have preferredselectivity of one of its receptors versus another. The inventionrelates to analogs based upon the polypeptide sequences identified inTables 1, 2, 3, and 4. All modified and unmodified variants of thesequences listed in Table 4 are contemplated as being part of theinvention. For instance, the sequence of Biotin-Bombesin is listed inTable 4 as Biotin-EQRLGNQWAVGHLM-NH2. Not only do analogs of the claimedinvention include biotinylated sequence above with an amidatedmethionine, but the analogs of the present invention also relate to theunmodified or modified polypeptide backbone EQRLGNQWAVGHLM as well asfunctional fragments thereof.

For purposes of interpreting polypeptide modifications throughout theapplication, please refer to the following legend:

-   Ac—Acylation-   p-Cl-dF=para-Chlorine, D-Phenylalanine-   4cl=Chlorinated Phenylalaine-   _(d)F=para-Chlorine, D-Phenylalanine-   _(d)R=D-Arginine-   _(d)Y=D-Tyrosine-   _(d)A=D-Alanine-   _(h)R=homoarginine-   pY=Phosphoroylated Tyrosine-   pS=Phosphoroylated Serine-   pE=Pyroglutamic acid-   PEG=Polyetheythlene Glycol-   PEG {number kD)}=Polyetheythlene Glycol with a molecular weight near    {number} in kilodaltons.-   Nle=Noraleucine-   N_(le)=Noraleucine-   Y_(m), =methoxy-tyrosine.-   Y_(M)=methoxy-tyrosine.-   K_(m)=methalyated-lysine.-   Aib=α-aminoisobutyric acid-   Abu=ALPHA-AMINOBUTYRIC ACID-   Gab=γ-aminobutyric acid;-   Dip=β,β-diphenyl-L-alanine;-   *=indicates cyclization between residues (lactam ring)-   dHis=D-His-   w=D-Tryptophan-   Dnp=di-nitro-phenol-   Mca=methoxycoumarin 4 acetic acid-   Sar=sarcosine-   Sta=statine-   Ste=Stearyl-   Pyr=pyroglutamic acid-   Fam=carboxyfluoresceine-   LC=—(NH₂—(CH₂)₅—C═O)—-   TAMRA=carboxytetramethylrhodamine-   T*=N-acetyl galactosamine labeled Thr-   NH₂=amidation of carboxy terminus-   Orn=ornithine-   K(W)=Trp residue which is coupled to the side chain of a Lys-   Y(OMe)=methylated Tyrosine-   Cit=citrulline-   C6=hexanoyl-   Nva=Norvaline    In some embodiments, analogs of the present invention (including any    polypeptide sequence identified in Tables 1, 2, 3, or 4) are either    be N-terminal acylated or an N-terminal free-amine. In some    embodiments, analogs of the present invention are either a    c-terminal amine or a c-terminal acid. These terminal groups do not    preclude additional solubilization and/or stabilization attachments    such a poly-ethylene glycol.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:1.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:1.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:2.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:2.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:3.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:3.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:4.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:4.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:5.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:5.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:6.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:6.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:7.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:7.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:8.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:8.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:9.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:9.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:10.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:10.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:11.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:11.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:12.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:12.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:13.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:13.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:14.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:14.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:15.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:15.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:16.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:16.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:17.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:17.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:18.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:18.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:19.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:19.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:20.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:20.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:21.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:21.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:22.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:22.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:23.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:23.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:24.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:24.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:25.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:25.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:26.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:26.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:27.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:27.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:28.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:28.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:29.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:29.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:30.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:30.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:31.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:31.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of SEQ ID NO:32.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of SEQ ID NO:32.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition comprising of any one or plurality ofpolypeptides chosen from: SEQ ID NO:1 through SEQ ID NO:64.

In some embodiments, the invention relates to any composition, kit, orpharmaceutical composition consisting of any one or plurality ofpolypeptides chosen from: SEQ ID NO:1 through SEQ ID NO:64.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

EXAMPLES Example 1 Chemical Scheme to Synthesize Polypeptides (notProphetic)

This example describes how the polypeptide analogs are manufactured. Thesequence of human vasoactive intestinal peptide (VIP) is given below,using the standard one-letter code for proteinogenic amino acidresidues. For purposes of interpretation “position 1” of the sequencebelow is the N-terminal histidine. Each amino acid residue is numberedin sequence from the N-terminal end of the polypeptide to the C-terminalHSDAVFTDNYTRLRKQMAVKKYLNSILN

Design.

A family of analogues will be prepared, each containing multiple a to β³replacements. Each β³-amino acid residue will bear the side chain of theα-amino acid indicated by the one-letter code. The analogues to beprepared are shown below; the positions indicated with lowercase lettersare those at which α-to-β³ replacement has occurred. In addition, then-terminal of these analogues may be optionally acetylated.

TABLE 1 VPAC2 selective analogs. EC50 values are the results ofcell-based cAMP assays.Values reported are in molar concentration. EC 50vpac1 EC50 vpac2 Identifier Sequence Seq ID (Molar) (Molar) LBT-V201Ac-HSDAVFTENYTKLRKQLA x KKY x ND l KKG g T Seq ID 1 9.088E−09 1.039E−07LBT-V202 Ac-HSDAVFTENYTKLRKQLAA z KY x NDLkKG g T Seq ID 2    >1E−69.629E−10 LBT-V203 Ac-HSDAVFTENYTKLRKQ x AA z KYL x DL k KGGT Seq ID 32.177E−07 1.804E−10 LBT-V204 Ac-HBDAVFTENYTKLRKQLAA z KY x NDL k KG g TSeq ID 4 5.163E−07 5.286E−10 LBT-V205 Ac-HBDA v FTENYTKLRKQLAA z KY xNDL k KG g T Seq ID 5    >1E−6 8.162E−10 LBT-V206 Ac-HBDA v FTE nYTKLRKQLAA z KY x NDL k KG g T Seq ID 6    >1E−6 3.472E−09 LBT-V207 Ac-Hx DAVFTENYTKLRKQLAA z KY x NDL k KG g T Seq ID 7    >1E−6 5.928E−10LBT-V208 Ac-H x DA x FTENYTKLRKQLAA z KY x NDL k KG g T Seq ID 8 1.33E−07  6.46E−10 LBT-V209 Ac-H x DA x FTE x YTKLRKQLAA z KY x NDL kKG g T Seq ID 9    >1E−6 5.579E−09 LBT-V210 Ac-H x DA v FTENYTKLRKQLAA zKY x NDL k KG g T Seq ID 10  1.42E−06 8.586E−10 LBT-V211 Ac-H x DA vFTENYTKLRKQ l AA z KY x NDL k KG g T Seq ID 11    >1E−6 1.258E−09LBT-V212 Ac-H x DA v FTENYTKLRK g LAA z KY x NDL k KG g T Seq ID 12 1.53E−06 3.515E−09 LBT-V213 Ac-H x DA v FTENYTKLR k QLAA z KY x NDL kKG g T Seq ID 13  2.93E−07 1.266E−09 LBT-V214 Ac-H x DA v FTENYTKL rKQLAA z KY x NDL k KG g T Seq ID 14    >1E−6 1.372E−09 LBT-V215 Ac-H xDA v FTENYTK l RKQLAA z KY x NDL k KG g T Seq ID 15  1.42E−06 1.334E−09LBT-V216 Ac-H x DA x FTE ny TKLRKQLAA z KY x NDL k KG g T Seq ID 169.318E−07 9.559E−08 LBT-V217 Ac-H x DA x FTE xy TKLRKQ l AA z KY x NDL kKG g T Seq ID 17 7.853E−07 1.166E−09 LBT-V218 Ac-H x DA x FTE xy TKLRK gLAA z KY x NDL k KG g T Seq ID 18    >1E−6 1.018E−09 LBT-V219 Ac-H x DAx FTE xy TKLR k QLAA z KY x NDL k KG g T Seq ID 19 4.614E−07    >1E−6LBT-V220 Ac-HSDAVFTENYTKLRKQ x AAK z YL x DLK k GGT Seq ID 20 9.515E−09 4.22E−10 LBT-V221 Ac-HSDAVFTDNYTRLRKQ x AAK z YL x SIK n KRY Seq ID 211.952E−10 7.845E−11 wherein Ac = acetyl; B = AIB.

In each of sequences above, at least one residue has been replaced by acyclic or heterocyclic β-amino acid residue. In some embodiments, basedupon the above sequences, X=ACPC, Z=APC; uncharged side chains replacedby ACPC, basic side chains replaced by APC, Protected β³-amino acids).Individual α/β amino acids (Fmoc on the backbone nitrogen andappropriate protecting groups on side chains, when necessary) will beobtained from commercial suppliers or prepared via reported methods.Each β³-peptide will be prepared manually by microwave-assisted Fmocsolid phase peptide synthesis resulting in a c-terminal amide, forexample Rink Amide resin. Coupling steps will be carried out with athree-fold excess of the appropriate protected α- or β³-amino acid,using HATU to mediate amide bond formation. Piperidine will be used forFmoc deprotection steps. Each peptide will be cleaved from resin bytreatment with 94:2.5:2.5 TFA/H2O/triisopropylsilane, precipitated byaddition of cold ethyl ether, and purified by reverse phase HPLC on aprep-C18 column using gradients between 0.1% TFA in water and 0.1% TFAin acetonitrile. The identity and purity of the final products will bedetermined by mass spectrometry and analytical HPLC, respectively.

One purpose of this study is to demonstrate that the analogs of theapplication may be designed to increase the half-life of the polypeptideas compared to the half-life of the naturally encoded protein byintroducing non-natural amino acid analogs that are resistant todegradation and/or induce an equivalent or increased bioactivity ascompared to the naturally encoded polypeptide sequence upon which theanalog is based or derived through the possible incorporation ofconformationally-constrained residues.

In-Vitro Cell Based Activity Assay

In Vitro Binding Assay 1: VIP analogues in table 1 were prepared inappropriate phosphate buffer was at pH of 7.5 was exposed to afunctional assay in parallel with wild-type VIP proteins. Divisionarrested cAMP Hunter cell lines (purchased from DiscoveRx) expressingVIPR1 and VIPR2 were plated into 96 or 384-well microplates for compoundprofiling according to the manufactures directions. Cells were allowedto adhere and recover overnight prior to compound addition. cAMPmodulation was determined using the DiscoveRx HitHunter cAMP XS+ assay.

For profiling compound in agonist mode, the cells were incubated in thepresence of compound at room temperature for 60 minutes. Cellsexpressing both VIPR1 and VIPR2 were exposed to serial dilutions (3 foldor 10 fold as appropriate) of wild-type VIP and separate samples of thesame type of cells were exposed to serial dilutions (3 fold or 10 foldas appropriate) of a VIP analogue to determine EC₅₀ values of theanalogue as compared to wild-type VIP. All dilutions were within therange of millimolar to picomolar in final compound concentration in eachwell. After appropriate compound incubation, assay signal was generatedthrough incubation with DiscoverX lysis cocktail according to themanufacturers standard protocol. Dose curves were plotted using GraphPadPrism. Percentage activity is calculated using the following formula:

% Activity=100%×(mean RLU of test sample−mean RLU of vehiclecontrol)/(mean RLU of MAX control−mean RLU of vehicle control).

In Vitro Competitive Binding Assay (Prophetic):

Binding assays: Membranes prepared from a stable VPAC2 cell line (suchas a CHO—S cell line stably expressing human VPAC2 receptor or fromcells transiently transfected with human VPAC1 or PAC1) will be used. Afilter binding assay will be performed using ¹²⁵I-labeled VIP for VPAC1and VPAC2 and ¹²⁵I-labeled PACAP-27 for PAC as the tracers. For thisassay, the solutions and equipment include:

Presoak solution: 0.5% Polyethyleneamine in Aqua destBuffer for flushing filter plates: 25 mM HEPES pH 7.4Blocking buffer: 25 mM HEPES pH 7.4; 0.2% protease free BSAAssay buffer: 25 mM HEPES pH 7.4; 0.5% protease free BSADilution and assay plate: PS-Microplate, U formFiltration Plate Multiscreen FB Opaque Plate; 1.0 mM Type B GlasfiberfilterIn order to prepare the filter plates, the presoak solution will beaspirated by vacuum filtration. The plates will be flushed twice with200 μL flush buffer. 200 μL blocking buffer will be added to the filterplate. The filter plate will then be incubated with 200 μL presoaksolution for 1 hour at room temperature. The assay plate will be filledwith 25 μL assay buffer, 25 μL membranes (2.5 μg) suspended in assaybuffer, 25 μL agonist in assay buffer, and 25 μL tracer (about 40000cpm) in assay buffer. The filled plate will be incubated for 1 hour withshaking. The transfer from assay plate to filter plate will beconducted. The blocking buffer will be aspirated by vacuum filtrationand washed two times with flush buffer. 90 μL will be transferred fromthe assay plate to the filter plate. The 90 μL transferred from assayplate will be aspirated and washed three times with 200 μL flush buffer.The plastic support is removed. It is dried for 1 hour at 60° C. 30 μLMicroscint will be added. The count will be performed based upon analogaffinity to VPAC1, VPAC2, or PAC1 receptors. IC₅₀ and EC₅₀ calculationswill be performed based upon affinity scoring.

Example 2 Structural Analysis of Helical Polypeptides (Prophetic)

This prophetic example describes how the polypeptide analogs of thisinvention may be characterized after manufacture through structuralconformational assays such as circular dichrosim (CD) and Nuclearmagnetic resonance (NMR).

Circular Dichroism Spectroscopy. Circular dichroism measurements will becarried out on an Aviv 202SF Circular Dichroism Spectrophotometer.Samples of each peptide will be prepared with a determined UV absorbancein the range of 0.1-1.0 at 280 nm in a pH buffered solution. Spectrawill be recorded in a 1 mm cell with a step size of 1 nm and anaveraging time of 5 sec. All spectra will be background correctedagainst buffer measured in the same cell. Thermal melts will be carriedout in 1-degree increments with an equilibration time of 2 min betweeneach temperature change. Thermal unfolding data will be fit to a simpletwo state folding model Shortle, D. Meeker, A. K. Freire, E.Biochemistry 1988, 27, 4761-4768) using GraphPad Prism.

Nuclear Magnetic Resonance: Structure elucidation of the proposedanalogs can also be accomplished based on analyses of heteronuclear NMRexperimental data. Global backbone structural information complementingthe local structure information provided by backbone chemical-shiftassignments can be obtained from nuclear Overhauser effect spectroscopy(NOESY) which yield atomic distance constraints together with residualdipolar coupling (RDC) experiments which provide orientation restraintinformation. Together, these techniques can be used to provide valuablestructural information regarding the positioning and alignment of theamino acids within the polypeptide analog. Samples of each peptide oranalog will be prepared with a determined UV absorbance in the range of0.1-1.0 at 280 nm in an appropriate pH buffered solution. Eachpreparation will then be used to determine chemical shifts using thesuite of multidimensional experiments, ie amide based backboneassignments HNCO and TOCSY, followed by conducting structure restraintexperiments, ie NOESY and RDC, using standard NMR equipment (i.e. BrukerNMR) and data analysis software (i.e. Talos+, SPARKY and Al NMR).Further structural insight can be ascertained by comparing the resultsof NMR experiments in the presence and absence of the intended bindingpartner.

One purpose of this study is to evidence that the conformation of theanalog is structurally constrained and that certain non-natural aminoacids have been incorporated in the synthesized peptide in theirpredicted location along a longitudinal axis of the polypeptide.

Example 3 In-Vitro Stability Analysis of Helical Polypeptides inSolution (not Prophetic)

This example describes how the metabolic stability of the polypeptideanalogs of this invention are characterized after manufacture throughassays such as a protease resistance assay.

In Vitro Stability Assay: Stock solutions of the both the naturallyoccurring peptides as well as peptide analogs are prepared at aconcentration of 250 μM (based on UV absorbance) in a 2% DMSO solutionof appropriate buffering conditions. Standard solution of proteinase Kin addition to other common animal proteases (i.e. Cathepsins, Trypsins,dipeptidyl peptidase IV and chymotrypsin) were prepared at the followingconcentrations in appropriate buffers separately: Protease K: 250 μg/ml,Chymotrypsin 25 μg/mL and DPP-IV 40 units/ml. For a Protease K assay,the following recipe was followed: 1 μL enzyme was added to 5 μL peptideanalog in 44 μL PBS. For a Chymotrypsin assay, the following recipe wasfollowed: 2 μL of enzyme was added to 5 μL of peptide analog in 43 μLPBS. For a DPP-IV assay, the following recipe was followed: 2 μL enzymewas added to 5 μL peptide analog in 18 μL PBS. All reactions (regardlessof enzyme or analog) were then allowed to proceed at room temperatureand quenched at the desired time point by addition of 10 μL of 1% TFA in99% Acetonitrile. 50 μL of the resulting quenched reaction are theninjected onto an analytical reverse phase HPLC, and the amount ofstarting peptide present quantified by integration of the appropriatechromatogram peak via absorbance at either 220 or 280 nm. Duplicatereactions are run for each time point. Half-lives are determined byfitting time dependent peptide concentration to an exponential decayusing GraphPad Prism. Samples for some time points will be analyzed bymass spectrometery, and the products observed are used to identify amidebonds cleaved in the course of the reaction. The relative stabilityenhancement is determined through the comparison of the various analogswith its naturally occurring peptide counterpart. Percent degradation isbe quantified by integration of peak areas related to undigested peptidepeaks and corrected for degradation in the absence of enzyme.

Similar protocols were followed to examine the stability of the VIPanalogs in simulated gastric fluid (SGF). SGF was prepared using thefollowing recipe: 2 g NaCl, 64 mg Pepsin

and 7 mL HCl were dissolved in 1 liter of deionized water. 45 μL of SGFwere mixed with 5 uL of peptide solution (stock concentration of 250 uM)and incubated together. At timepoints, 0, 1, 2, 5, 10, 15, 20 minutesreactions were quenched with 10 uL 1M Tris pH 8.0 to stop the digestion.Final samples were injected onto an analytical reverse phase HPLC, andthe amount of starting peptide present quantified by integration of theappropriate chromatogram peak via absorbance at either 220 or 280 nm.Duplicate reactions are run for each time point. Half-lives aredetermined by fitting time dependent peptide concentration to anexponential decay using GraphPad Prism. Samples for some time pointswere analyzed by mass spectrometery, and the products observed were usedto identify amide bonds cleaved in the course of the reaction. Therelative stability enhancement is determined through the comparison ofthe various analogs with its naturally occurring peptide counterpart.Percent degradation is be quantified by integration of peak areasrelated to undigested peptide peaks and corrected for degradation in theabsence of enzyme.

FIG. 1: Representative in-vitro protease stability of VPAC-2 selectiveanalog (LBT-V208) compared to the native VIP as well as the VPAC2selective agonist Ro 25-1553. It is noteworthy that the stability isgreater for the non-lactam containing LBT-V208 as compared to Ro25-1553, which contains one lactam bridge between lysine and asparticacid residues.

FIG. 3: Representative in-vitro protease stability of VPAC-1 selectiveanalog (LBT-V101) compared to the native VIP.

Ex-vivo Stability Assay (prophetic): To investigate the plasma stabilityof the analogs, both the naturally occurring peptide as well as theanalogs will be prepared at a concentration of 100 μM (based on UVabsorbance) in appropriate buffer. 50 uL aliquots of animal plasma (i.e.rodent, canine, primate) are then spiked with the analog or thenaturally occurring peptide. The reaction will be allowed to proceed atroom temperature and quenched at the desired time point by addition ofan equivalent volume of 1% TFA in Acetonitrile and diluted 1-10 foldwith PBS. This solution is then passed over a C18 solid phase extractioncolumn (eg. Sigma TPSC18) to further isolate the peptide or analog forsubsequent LC/MS analysis by removal of unrelated lipids and plasmaproteins. The analogs are then eluted from the C18 column by adding 1-3column volumes of between 20 and 50% acetonitrile in 0.1% formic acid,collected and concentrated for subsequent analysis. Approximately 10 μLof the concentrated quenched reaction will be injected onto ananalytical reverse phase HPLC, and the amount of starting peptidepresent quantified by integration of the appropriate chromatogram peakvia absorbance at either 220 or 280 nm. Duplicate reactions will be runfor each time point. Half-lives will be determined by fitting timedependent peptide concentration to an exponential decay using GraphPadPrism. Samples for some time points will be analyzed by massspectrometery, and the products observed will be used to identify amidebonds cleaved in the course of the reaction. The relative stabilityenhancement will be determined through the comparison of the variousanalogs with its naturally occurring peptide counterpart.

Microsome Stability Analysis of Analogs Polypeptides in Solution(prophetic): A reaction mixture, minus NADPH, should be prepared asdescribed below. Approximately 1 milligram of test compound originallyin powdered form is suspended in DMSO prior to addition to a reactionmixture. The test compound was added into the reaction mixture (0.5mg/mL human liver microsomes; 100 mM potassium phosphate; 5 mM Magnesiumchloride) at a final concentration of 1 μM. An aliquot of the reactionmixture (without cofactor) was incubated in a shaking water bath at 37°C. for 3 minutes. The control compound, testosterone, should be runsimultaneously with the test compound in a separate reaction. Thereaction is initiated by the addition of NADPH cofactor (1 mM NADPH),and the mixture was then incubated in a shaking water bath at 37° C.Aliquots (100 μL) are withdrawn at 0, 10, 20, 30, and 60 minutes for thetest compound and 0, 10, 30, and 60 minutes for testosterone. Testcompound and testosterone samples are immediately combined with 400 μLof ice-cold 50/50 acetonitrile/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. The samples are then mixedand centrifuged to precipitate microsomal proteins. Testosterone samplesare assayed via LC-MS/MS using electrospray ionization on an appropriatetriple-quadropole mass spectrometer according to the manufacturer'sinstructions. A thermo BDS Hypersil C18 column (30×2.0 mm; 3 μm) can beused for chromatography at 300 μL/minute with an aqueous reservoir of90% water and 10% buffer and an organic reservoir of 90% acetonitrilewith 10% buffer (each 25 mM ammonium formate buffer at pH of 3.5). Testcompound samples are also analyzed by using LC-MS/MS mass spectrometer.The peak area response ratio to internal standard (PARR) of thecompounds at 10, 20, 30, and 60 minutes are then compared to the PARR attime 0 to determine the percent of test compound remaining at eachtimepoint. After the final time point, fluorimetry is used to confirmthe addition of NADPH to the reaction mixture. Half-life was normalizedof control using internal acceptance criteria. Half-life was calculatedbased upon a t1/2=0.693/k, where k is the elimination rate constantbased upon the slope of the plot of natural logarithm percent remainingversus incubation time. Intrinsic clearance (CL_(int)) was calculatedbased upon CL_(int)=k/P, where k is the elimination rate constant and Pis the protein concentration in the incubation.

In Vivo Stability Assay (not prophetic): To investigate the in vivostability of the analogs, both the naturally occurring peptide as wellas the analogs were administered to male C57BL/6 mice by IV, SC and POroutes at concentrations ranging from 0.001 to 50 mg/kg and bloodspecimens drawn at periodic time-points for up to 24 hourspost-injection. Specific dose concentrations used include 50 nmol/kg,100 nmol/kg 250 nmol/kg 500 nmol/kg and 1000 nm/kg. Specific time-pointsused to perform these studies include: 0.5, 1, 2, 5, 8 and 20 hoursafter initial delivery. Levels of intact compound in 10 μL of freshlycollected plasma was determined by injection onto an analytical reversephase HPLC after following the solid phase extraction procedurediscussed previously. The amount of starting peptide present quantifiedby integration of the appropriate chromatogram peak via absorbance ateither 220 or 280 nm or other means of measuring the presence or absenceof fully intact analog as described herein. Samples were analyzed usingMultiple Reaction Monitoring (MRM) mass spectrometry techniques whereinthe amount of full-length (as judged by calculated molecular weight)analog was determined by integration of the data resulting from the MRManalysis and compared against a standard curve of known amounts ofanalog under the same conditions. This analysis technique also allowsthe examination of the in-vivo metabolites by determination of fragmentmolecular weights. Area-under-the-curve calculations were performedusing Graphpad Prism on the MRM data resulting from LC/MS-MS analysis,thus resulting in a relative concentration value for each data pointcollected. The relative stability enhancement was determined through thecomparison of the relative concentration values of the various analogswith its naturally occurring peptide counterpart.

FIG. 4. Representative pharmacokinetic study of seqID: 218 viasubcutaneous administration in C57 BL/6 male mice. Analysis conductedvia LC/MS/MS techniques to confirm presence of target molecule. Pointsrepresent AUC values for each time point described in the x-axis.

Determination of Oral Bioavailability (not prophetic): The oralbioavailability was evaluated by comparative analysis betweenIntraduodenal and Intravenous administration of the analogs. Eachcompound/test article was first converted to an appropriate salt formand dissolved in a combination of biocompatible acids (e.g. citric acid,citrate, taurodeoxycholic acid, stearic acid, etc.) along with anoptional permeation enhancer (e.g. DL-Lauroylcarnitine, sodium laurylsulfate, Acetylated monoglycerides, sucrose, chitosan, tri-methylchitosan, etc) or buffer (e.g. PBS, sodium bicarbonate, etc). Chitosanwas purchased from TCI chemicals with a range of viscosities (5 mPa to527 mPa) each with an average degree of deacetylation between (80-90%).Trimethyl chitosan (TMC) was prepared based on these chitosan samplesaccording to the two-step synthesis protocol described by Sieval et. al.in Carbohydrate Polymers 36 (1998) 157-165. Specifically, A mixture of 2g of sieved chitosan (˜80-90% deacetylated), 4.8 g of sodium iodide, 11ml of a 15% aqueous sodium hydroxide solution and 11.5 ml of methyliodide in 80 ml of 1-methyl-2-pyrrolidinone was stirred on a water bathof 60° C. for 1 h (Le Dung et al., 1994). Special care was taken to keepthe methyl iodide in the reaction mixture by using a Liebig condenser.The product was precipitated using ethanol and thereafter isolated bycentrifugation. The N-trimethyl chitosan iodide obtained after thisfirst step was washed twice with ether to remove the ethanol. It wasdissolved in 80 ml of 1-methyl-2-pyrrolidinone and heated to 60° C.,thus removing most of the absorbed ether. Subsequently, 4.8 g of NaI, 11ml of 15% NaOH solution and 7 ml of methyl iodide were added with rapidstirring and the mixture was heated on a water bath at 60° C. for 30min. An additional 2 ml of methyl iodide and 0.6 g of NaOH pellets wereadded and the stirring was continued for 1 h. The product, prepared asdescribed above, was dissolved in 40 ml of a 10% NaCl aqueous solution,instead of HCl, to exchange the iodide. The polymer was precipitatedwith ethanol, isolated by centrifugation and thoroughly washed withethanol and ether. In vacua drying yielded a white, water-solublepowder. Subsequently TMC solutions for each of the initial chitosan(varying viscosities ˜5 to ˜500 mPa) starting materials ranging inconcentrations from 0.1%-20% TMC in biologically acceptable buffers(PBS, Sodium bicarbonate, etc) were prepared. Degree of tri-methylationwas confirmed via NMR techniques.

FIG. 10: Representative NMR spectra of TMC (in DCl) prepared fromchitosan with viscosity of 5 mPa. The large peak observed at 3.65 ppm isassigned to the tri-methylated state of chitosan, indicating completetri-methylation.

Subsequently, intraduodenal (ID) catheterized male C57/BL6 mice, 8 weeksold (Charles River Labs) are quarantined for at least one day and havecontinuous access to food and water. A single dose of the appropriateformulated analog is then administered via catheter to each mouse.Single doses of 1000 nmol/kg (seq ids 208, 218 and 101) were dissolvedin 5% TMC in PBS (pH 7.5) and delivered via intraduodenal catheterinfusion.

Blood samples were collected via tail vein at the following time points:0 h, 0.75 h, 1.5 h, 3 h. Samples were kept in EDTA containing (10 uL of50 mM) microtainer tubes under subambient temperature (4° C.) beforethey are processed. Blood samples were centrifuged (10,000 rpm for 5minutes) and plasma samples were flash frozen with liquid nitrogen andsubsequently stored in a −20° C. freezer until analyzed for analoglevels. Analog levels in the plasma will be analyzed using the followingprotocol for direct plasma precipitation.

The in vivo plasma samples are then prepared in 0.5 mL microfuge tube,by adding, 10 μL of test plasma to 50 μL of cold methanol with 0.1%formic acid followed by vortexing for 10 minutes at 4° C. Samples arethen centrifuged for 3 minutes, when the supernatant is decanted into anew tube and lyophilized. The dried supernatant is then resuspended in12 μL of 20% acetonitrile, 0.1% formic acid. 10 μL of this final samplewill then be injected into the LC/MS-MS for analysis. The preparedsample will be injected onto a reverse phase column (Phenomonex 3.0×50mm) using a mobile phase of 20% CH₃OH, 0.1% formic-35% CH₃OH, 0.1%formic acid for LC/MS-MS analysis. The run time will be about 10 minutesat a flow rate of about 400 μL/minutes. The Area Under the Curve (AUC)will be calculated using the linear trapezoidal rule from t=0 to thelast plasma concentration sampling time tx (see Handbook of BasicPharmacokinetics, Wolfgang A. Ritschel and Gregory L. Kearns, 5th ed,1999). AUC⁰-tx=.SIGMA.⁰-n((C_(n)+C_(n)+1)/2))(t_(n)+1−t_(n)) {in(μg/mL)h}

One purpose of this study is to evidence that the analog is moreresistant to peptidases as compared to the resistance ofsimilarly-structured, naturally occurring polypeptides upon which thestructure of the analog is based or derived. The results may show that,when treated with the same proteolytic enzymes or in vivo biologicalconditions, the analogs of the invention will resist degradation, becomesubstantially more bioavailable and have longer half-lives thansimilarly-structured, naturally occurring polypeptides upon which thestructure of the analog is based or derived.

Example 4 In-Vivo Functional Analysis of VPAC2 Selective Polypeptides(Prophetic)

This prophetic example describes the function of polypeptide analogs ofthis invention may be characterized after manufacture through assaysthat measure bioactivity of the analogs when exposed to tissue cultureor when administered to an animal model of one of the following humandisease states: COPD, pulmonary hypertension, primary arterialhypertension, pulmonary hypertension associated to post-ventricularseptal defect, idiopathic pulmonary fibrosis, idiopathic pulmonaryarterial hypertension, CREST syndrome—Calcinosis; Raynaud's disease;loss of muscle control of the Esophagus; Sclerodactyly; Telangiectasia,Acute respiratory distress, congestive heart failure, chronic obstructedpulmonary disorder, asthma, chronic obstructive pulmonary disease,sarcoidosis, small lung cell cancer, exocrine pancreatic tumors,colorectal carcinoma, gastric carcinoma, hepatocellular carcinoma,esophageal carcinoma, renal cell carcinoma, prostate carcinoma, urinarybladder carcinoma, liver carcinoma, ductal pancreatic cancer, breastcarcinoma, ovarian carcinoma, non-hodgkin's lymphoma, meningioma, GEPtumors (differentiated and undifferentiated), pituitary adenoma,endometrial cancer, astrocytoma, giloblastoma, non-small cell lungcancer, pancreatic cancer, melanoma, renal cancer, neuroblastoma,leukima and prostate cancer, autoimmune disease, inflammatory disease,sepsis, Hirschsprung's Disease, sexual dysfunction, erectiledysfunction, Parkinson's disease, Alzheimer's disease, circadian rhythmdysfunction, pain, colorectal cancer, hepatocellular cancer, elevatedblood pressure levels, elevated blood glucose levels, elevated bloodpressure levels, hyperglycemia, diabetes, insulin resistance, metabolicacidosis, obesity, Type I diabetes, Type II diabetes Multiple Sclerosis,osteoporosis, Sjogren's syndrome, pancreatitis, uveoretinitis,osteoporosis, female sexual dysfunction and diabetic nephropathy.

In Vivo Efficacy in Animal Models: To determine the activity of analogsof the invention in vivo as compared to the naturally occurringpolypeptides upon which the analogs are derived, the analogs will beadministered alone (IP, IV, SC, PO, by inhalation or nasal routes) or incombination with known active agent to monitor the above-mentioneddisease states. Secretin family analogs alone or in combination withsub-optimal doses of relevant active agents for specific indications ordisease states will be, for example, administered to an appropriateanimal model mice (8-10 days after injection/day 1 of experiment) bytail vein or IP routes at doses ranging from 0.0001 mg/kg to 50 mg/kgfor 1 to 21 days. Optionally, the mice will be assayed throughout theexperiment with a selection marker relevant to the particular studiesdisease state every other day and survival monitored daily for theduration of the experiment. Expired mice will be optionally subjected tonecropsy at the end of the experiment. These in vivo tests optionallygenerate preliminary pharmacokinetic, pharmacodynamic and toxicologydata.

Adjuvant-Induced Arthritis in Rats: Adjuvant induced arthritis (“AIA”)is an animal model useful in the study of rheumatoid arthritis (“RA”),which is induced by injecting M. tuberculosis in the base of the tail ofLewis rats. Between 10 and 15 days following injection, animals developa severe, progressive arthritis.

Generally, analogs will be tested for their ability to alter hind pawswelling and bone damage resulting from adjuvant induced edema in rats.To quantitate the inhibition of hind paw swelling resulting from AIA,two phases of inflammation have been defined: (1) the primary andsecondary injected hind paw, and (2) the secondary uninjected hind paw,which generally begins developing about eleven days from the inductionof inflammation in the injected paw. Reduction of the latter type ofinflammation is an indication of immunosuppressive activity. Cf. Chang,Arth. Rheum., 20, 1135-1141 (1977).

Using an animal model of RA, such as AIA, enables one to study thecellular events involved in the early stages of the disease. CD44expression on macrophages and lymphocytes is up regulated during theearly development of adjuvant arthritis, whereas LFA 1 expression is upregulated later in the development of the disease. Understanding theinteractions between adhesion molecules and endothelium at the earlieststages of adjuvant arthritis could lead to significant advances in themethods used in the treatment of RA.

Collagen Induced Arthritis in Rats: To determine the efficacy of arepresentative analog of this invention administered by po, iv, sc ornasal dosing according to bid or qd schedules (Days (−1)-20) forinhibition of the inflammation, cartilage destruction and boneresorption that occurs in developing type II collagen arthritis in rats.

Animals: Female Lewis rats (Harlan), weighing 125-150 g on arrival.(inject subtotal of rats with collagen to get responders on days 10, 11,12 for 6 groups of 10). The animals (a group for arthritis, a group fornormal control), housed 4-5/cage, will be acclimated for 4-8 days. Theanimals will be dosed from about po1 mg/kg bid to po100 mg/kg bid.

Materials: Peptides or analogs in vehicle, Type II collagen, Freund'sincomplete adjuvant, methotrexate (Sigma)

General Study Design: Dosing initiated on day minus 1. The acclimatedanimals will be anesthetized with isoflurane and given collageninjections (D0). On day 6 they will be anesthetized again for the secondcollagen injection. Collagen is prepared by making a 4 mg/mL solution in0.01 N acetic acid. Equal volumes of collagen and Freund's incompleteadjuvant, will be emulsified by hand mixing until a bead of thismaterial held its form when placed in water. Each animal will receive300 uL of the mixture each time spread over 3 sites on back. Caliperingof normal (pre-disease) right and left ankle joints are to be doneapproximately one ay prior to the expected days on onset of disease.

Rats will be weighed on days (−) 1, 6, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, and 20 of the study and caliper measurements of ankles takenevery day beginning on day 9. Final body weights will be taken on day20. After final body weight measurement, animals are to be anesthetizedfor terminal plasma collection and then euthanization. Both hind pawsand knees will be removed. Hind paws will be weighed, placed (withknees) in formalin and then processed for microscopy.

Processing of Joints: Following 1-2 days in fixative and then 4-5 daysin decalcifier, the ankle joints will be cut in half longitudinally,knees will be cut in half in the frontal plane, processed, embedded,sectioned and stained with toluidine blue.

Induction of Colitis in HLA-B27 Rats: The efficacy of the analogs of thepresent invention in reversing colitis can be determined in HLA-B27transgenic rats. HLA-B27 transgenic rats have been utilized as an animalmodel of Inflammatory Bowel Disease which mimics Crohn's Disease inhumans. The rats overexpress the human MHC class I HLA-B27 heavy chainand beta-2 microglobulin proteins, which induces a variety of autoimmunediseases that include inflammation of the colon.

The therapeutic effect of the analogs described in this invention interms of resolving colitis can be evaluated in HLA-B27 transgenic rats.Diseased rats will be dosed subcutaneously with 0.001-100 mg/kg of asingle analog of this invention once or twice a day for 16 days or onceper week for two weeks.

Disease Activity Index (DAI) scores will be used to determine theefficacy of each analog as compared to rats dosed with vehicle. Inaddition, fecal consistency and FOB scores for both rats dosed withanalogs will be statistically compared to the vehicle group.

Induction of Colitis: 1-20 HLA-B27 (6-9 weeks old) transgenic rats willbe acclimated in animal facility for 10 weeks Animal bedding will bemixed from different cages once a week to control for a “dirty”environmental flora.

Treatments: Rats are to be enrolled and randomized into four groups(n=5) based on weight and DAI scores (FC.gtoreq.3, FOB.gtoreq.2). Theexperimental groups will be dosed subcutaneously with an analog0.001-100 mg/kg once or twice a day for 16 days or once per week for twoweeks and terminated at trough. The control groups include avehicle-treated group and a GG5/3 (mouse anti-rat alpha-4 integrinantibody) positive control group dosed subcutaneously at 10 mg/kg (5mL/kg) on d0, d3, and d6 and terminated at trough on d8. Fresh analogand vehicle treatments are to be formulated in advance of treatment.

Endpoint Read-outs: Disease Activity Index scores, Fecal Consistencytest and Fecal Occult Blood test, are to be taken 4 times a week togenerate in-life clinical scores. The primary read-out for the study isa histopathological analysis of cecum, proximal colon, mid-colon, anddistal colon. An IBD scoring system was applied (Table H2). TABLE H2 IBDScoring System Multiple Endpoints A Destruction of epithelium and glandsB Dilatation of glandular crypts C Depletion and loss of goblet cells DInflammatory cell infiltrates E Edema F Vascular congestion G CryptAbscesses H Atrophia.

Primary Arterial Hypertension animal model: 36 adult male Sprague-Dawleyrats (300-350 g in body weight are to be randomized for treatment 22days after a s.c. injection of saline or 60 mg/kg monocrotaline(MCT)(Sigma-Aldrich) to induce pulmonary hypertension. In addition to agroup of untreated rats, the experimental groups included rats that willreceive daily, weekly or monthly delivery of a secretin analog at anappropriate dose of (0.001-50 mg/kg or the delivery vehicle alone. OnDay 22 a carotid/femoral artery will be accessed for arterial bloodgases (systemic blood pressure can be monitored as well). Thoracotomywould then be performed and right ventricle catheterized with a Millarcatheter (or other appropriate catheter), which will be advanced to thepulmonary artery. Animals will have anesthesia induced and maintained onisoflurane throughout the experiment. Rats will be intubated prior tosurgical procedures. Hemodynamic measurements such as Pulmonary arterialpressure, systemic blood pressure (SAP, DAP, MAP) and heart rate are tobe collected continuously via a Gould-Ponemah physiograph. Statisticalanalysis will be performed on all hemodynamic data. Arterial bloodsamples will be collected at protocol specified time points (up to 8time points) for analysis of drug concentration and/or arterial bloodgases. Animals will euthanized after 30 minutes and lungs will becollected and snap frozen for subsequent analyzed of levels of drug.Animals are to be clinically observed once daily with body weightmeasured weekly.

Another model of PAH requires chronic exposure to a hypoxic environment(e.g. 10% O₂) and optionally a VEGF inhibitor to further exacerbate thecondition. Mice will be housed in this environment for up to three weekswith ad lib access to food and water. In some cases, the mice willreceive implantable telemetry devices (e.g. DSI-HD-S21) capable ofroutinely monitoring both systemic and pulmonary arterial bloodpressures along with body temperature and other relevant biometrics. Theexperimental protocol entails a 1-8 week preconditioning housing stepwhere mice are housed in the hypoxic environment before drugs areadministered. In some cases, the VEGF-inhibitor as well as the testarticles are dosed on a regular schedule during the preconditioningstep. Once begun, the study would last between 7-28 days wherein themice will be evaluated between 2-14 times a week using the telemetrysystem to acquire real time in vivo data. Comparison between theuntreated, analog treated and positive control (current approved orclinical stage PAH therapeutics) arms consist of the determination ofrelative percent change in arterial blood flow, arterial blood pressure,overall survival along with pathology and histology analysis. Pathologyand histology studies will be conducted post-mortem, post perfusion andsubsequent sectioning and staining (e.g. H&E) following standardbiological sample preparation procedures known to those skilled in theart. Comparative analysis is to be performed based on the resultingslides, including average artery diameter and density of inflammatorymarkers near the blood vessels.

While some embodiments of the invention have been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

Parkinson's Disease (PD) Model (not prophetic): Male C57/BL6 mice, 8-12wold, housed in the IACUC approved housing were administered an analogdescribed herein as well as a pharmaceutically acceptable carrier forfive days at a dose between (0.1 nM and 100 nM). Thereafter, SPC andTregs from the mice are harvested and then transferred to another set ofMPTP intoxicated mice using the following protocol.

Immediately following the final dose, mice were sacrificed, andsingle-cell suspensions were prepared from inguinal lymph nodes andspleens. CD4+ T cell populations from spleens and lymph nodes wereenriched by negative selection with CD4-enrichment columns (R&D Systems,Minneapolis, Minn.), followed by CD25-PE positive selection withAutoMACS (Miltenyi Biotec, Auburn, Calif.). As determined by flowcytometric analysis, populations of Tregs and Teffs were consistently0.95% pure using this method (12). T cells were cultured in completeRPMI 1640 (RPMI 1640 [Invitrogen, Carlsbad, Calif.] supplemented with10% FBS, 2 mM L-gluta-mine, 25 mM HEPES, 1 mM sodium pyruvate, 13nonessential amino acids, 55 nM 2-ME, 100 Um′ penicillin, and 100 mg/mlstreptomycin [Mediatech, Manassas, Va.]) in the presence of anti-CD3(145-2C11; BD Pharmingen, San Diego, Calif.), 4YSyn, or N-4YSyn.Proliferation and inhibition assays were performed, as described (3,10). MPTP-intoxicated mice received an i.v. tail injection of 5.3×10̂7freshly isolated SPCs or 1.3×10̂6 freshly enriched Tregs in 0.25 ml HBSS.Each Th subset was harvested, and 10̂3-10̂6 T cells from each subset weretransferred to separate recipient groups. For stimulation of cytokineproduction, Th subsets were stimulated with 20 ng/ml PMA and 1 mMionomycin (Sigma-Aldrich) for 5 h, cells were washed, media werereplaced, and supernatants were collected 24 h later for analysis. (JImmunol 2010; 184:2261-2271). Recipient mice were then followed andsacrificed, their brains were excised and stained with appropriatemarkers for microglia cell identification according to the followingprotocol:

Mice were postmortem transcardially perfused with PBS, followed by 4%para-formaldehyde (Sigma-Aldrich). Frozen midbrain sections (30 mm) wereimmunostained for Mac-1 (CD11b, 1:1000; Serotec, Raleigh, N.C.).Fluorojade C (FJ-C) staining (Millipore, Billerica, Mass.) was performedon adjacent sections, according to the manufacturer's protocol, toassess de-generating neurons and was quantified using ImageJ. Overall,dopaminergic neuron survival was assessed 7 d following MPTPintoxication and resolution of cell death processes with polyclonal Absto mouse tyrosine hydroxylase (TH; 1:1000; EMD Chemicals/Calbiochem, SanDiego, Calif.) and were counterstained for Niss1 substance by thioninstaining Total numbers of Mac-1+ cells, CD4+ T cells, and TH- andNiss1-stained neurons in the SN were estimated by stereo-logicalanalysis with Stereo Investigator software (MBF Bioscience, Williston,Vt.), using the optical fractionator module. Quantitation of striatal TH(1:500; EMD Chemicals/Calbiochem, Gibbstown, N.J.) was performed bydensitometric analysis. Adjacent midbrain sections were immunostainedfor CD4 (clone RM4-5, 1:200, BD Pharmingen). Sections were incubated instreptavidin-HRP solution (ABC Elite vector kit, Vector Laboratories,Burlingame, Calif.) and color developed using a generation systemconsisting of diaminobenzidine (DAB) chromogen (Sigma-Aldrich). (JImmunol 2010; 184:2261-2271).

Comparisons between treated and untreated brain sections were conductedand evaluated for neuroprotection and neuroregeneration. Brain sectionimages were analyzed for stain density and location to arrive at astatistical performance evaluation.FIG. 5. Adoptive transfer of splenocytes from drug-treated donors isneuroprotective in vivo. A. Photomicrographs of TH+/Niss1+ neurons inthe SN and TH+ striatal termini in mice treated with PBS, MPTP, or MPTPfollowed by adoptive transfer of splenocytes from either VIP, VPAC1, orVPAC2-treated mice. Sections were immunostained with anti-TH andHRP-conjugated secondary Ab and visualized with DAB. SN sections werecounterstained with thionin. B. Total number of surviving dopaminergicneurons (TH+/Niss1+) and nondopaminergic neurons (TH−/Niss1+) in the SNfollowing MPTP treatment and adoptive transfer. Percentages of spareddopaminergic neurons are included for each treatment. C. Relative THdensitometry of total dopaminergic termini in the striatum. B and C.Differences in means (±SEM, n=8 mice per group) were determined whereP<0.05 compared with groups treated with PBS (a) and MPTP (b). VIP,VPAC1 (seq id 101) and VPAC2 (seq id 208) arms received 15 ug/day/5 days

Direct administration of analog performance in a PD model. 70 MaleC57/BL6 mice, 8-12w old, housed in the IACUC approved housing across 10arms (n=7 per arm) were administered an analog described herein as wellas a pharmaceutically acceptable carrier for five days at a dose between(0.1 nM and 100 nM) or are administered a control (PBS or VIP) dose.Thereafter, all mice were intoxicated by MPTP delivery mice and analyzedaccording to the following protocol. 1 day following the final dose, thesame mice were administered MPTP (16 mg/kg) and followed for additionalanalysis. Two days after MPTP delivery a subset of mice (arms 1-5) weresacrificed according the procedures described above. These mice werethen evaluated for Mac-1+ expression levels. Five days after that (totalof 7 days post MPTP treatment), the remaining mice (arms 6-10) were alsosacrificed according the procedures described above and evaluated forneurological damage Immunological, pathological and other relativeanalysis were performed as described above. Comparisons between treatedand untreated brain sections were conducted and evaluated forneuroprotection and neuroregeneration. Brain section images can beanalyzed for stain density and location to arrive at a statisticalperformance evaluation.

FIG. 6. Average Mac-1+ expression for each of the arm of the directadministration study. Mice were administered 15 ug VIP, VPAC1 (Seq id101), VPAC2 (seq id 218) per day for 5 days prior to MPTP challenge.Mice were sacrificed and Mac-1+ levels were determined 2 days later.FIG. 7. Adoptive transfer of splenocytes from drug-treated donors isneuroprotective in vivo. Total number of surviving dopaminergic neurons(TH+/Niss1+) and nondopaminergic neurons (TH−/Niss1+) in the SNfollowing direct administration and MPTP treatment. Percentages ofspared dopaminergic neurons are included for each treatment. Differencesin means (±SEM, n=8 mice per group) were determined where P<0.05compared with groups treated with PBS (a) and MPTP (b). VIP, VPAC1 (seqid 101) and VPAC2 (seq id 218) arms received 15 ug/day/5 days.To assess whether VIP, Seq ID 101, Seq ID 218 and PBS treatedsplenocytes (SPC) suppress effector T cell proliferative responses, weevaluated SPC co-cultures from VIP-treated donors for theirproliferative capacity in the presence of either anti-CD3. At a 1:1ratio of SPC to VIP SPC, proliferation to anti-CD3 stimulation weresuppressed and diminished in a dose dependent fashion with thediminution of VIP SPC number. We hypothesized that Treg functionstimulated by VIP, seq id 101, seq id 218. To test this hypothesis, weevaluated CD4+CD25+CD62Llow Treg isolated from naïve and VIP-treatedmice for their capacity to inhibit CD3-mediated proliferation ofCD4+CD25− naïve T cells. VIP Treg showed increased functional capacityto suppress T cell proliferation compared with naïve Treg showing aconsistent or greater inhibition of proliferation. VIP and seq id 101,seq id 218 Treg showed enhanced suppressive capacity compared to PBSTreg populations, with greater inhibition versus naïve Treg at a 1:1Treg to responder ratio. These data suggested that seq id 101 and seq id218 enhances T cell regulatory function.FIG. 8. Inhibition assay to assess the suppressive function of Tregisolated from each donor group which includes VPAC1 (Seq ID 101), VPAC2(Seq ID 218), VIP and PBS (no treatment) on proliferation of anti-CD3stimulated naïve CD4+CD25− T cells. injections given daily/5 days/i.p.,VIP 15 ug, VPAC1 and VPAC2 300 ug.

In-vivo Cancer Model (prophetic): Female athymic BALByc nude mice, 4-5weeks old, will be housed in filter-top cages in a pathogen free,temperature-controlled, laminar-flow, filtered-air, isolated room andwill be exposed to light from 7:00 a.m. to 7:00 p.m. Cells from theappropriate tumor type will be injected subcutaneously into the rightflank of each mouse. Four experimental groups, of four mice each, threeof which will receive VIP and/or an analog of VIP (1.0, 5.0, or 10mg/day) in PBS; as a control, the fourth will receive only PBS. Allsolutions will be infused for 8 weeks, beginning 1 week after injectionof the cells, and delivered by i.v., i.p., subc., i.m. injection orosmotic pumps placed aseptically under the skin of the back of the mice.The pump will release its contents at a rate of 0.5 ml/h for a durationof 2 weeks. The spent pumps will be removed every 2 weeks, and newpumps, containing fresh solutions, will be implanted with knowntechniques; this procedure will be repeated three times. Aftertreatment, the tumors will be measured with calipers, and the mice willbe weighed weekly for 8 weeks. Tumor volume will be calculated for anellipsoid as (maximal length)×(maximal height)×(maximal width)×(π/6). Onthe last day of the experiment, blood will be sampled from either theretroorbital plexus or tail vein into chilled heparin-containing tubesrinsed with 0.05% NaEDTA and containing three protease inhibitors, 10mg/ml soybean trypsin inhibitor, 100 TIU/ml aprotinin, and 10 mg/mlphosphamidon), as well as 0.1 mM IBMX for measurement of plasma VIP andcAMP levels. The mice will then euthanized. The tumors will be excised,weighed, and frozen in liquid nitrogen for subsequent extraction (inmethanol) and for measurement of protein content by known techniques; aportion of the tumor will be fixed in 10% neutral buffered formalin formorphologic examination.

One purpose of these studies is to evidence that the analogs are capableof producing the desired biological, biochemical, diagnostic, medicinaland/or therapeutic outcome in a living animal.

Example 5 Chemical Scheme to Synthesize Polypeptides (not Prophetic)

This example describes how the polypeptide analogs are manufactured. Thesequence of human vasoactive intestinal peptide (VIP) is given below,using the standard one-letter code for proteinogenic amino acidresidues. For purposes of interpretation “position 1” of the sequencebelow is the N-terminal histidine. Each amino acid residue is numberedin sequence from the N-terminal end of the polypeptide to the C-terminal

HSDAVFTDNYTRLRKQMAVKKYLNSILN

Design.

A family of analogues will be prepared, each containing multiple a to β³replacements. Each β³-amino acid residue will bear the side chain of theα-amino acid indicated by the one-letter code. The analogues to beprepared are shown below; the positions indicated with lowercase lettersare those at which α-to-β³ replacement has occurred. In addition, then-terminal of these analogues may be optionally acetylated.

TABLE 2 VPAC1 selective analogs. EC50 values are the results ofcell-based cAMP assays. Values reported are in molar concentration. EC50vpac1 EC50 vpac2 Identifier Sequence Seq ID (Molar) (Molar) LBT-V101HSDAVFTDNY t RLR k QLA v KKY l NAI l N Seq ID 22  2.17E−09 0.008262LBT-V102 H x DAVFTDNY t RLR k QLA v KKY l NAI l N Seq ID 23 5.658E−09 4.90E−06 LBT-V103 H x DA x FTDNY t RLR k QLA v KKY l NAI l N Seq ID 243.575E−10 7.964E−08 LBT-V104 H x DA v FTDNY t RLR k QLA v KKY l NAI l NSeq ID 25 4.426E−09 2.624E−07 LBT-V105 H s DA v FTDNY t RLR k QLA v KKYl NAI l N Seq ID 26  6.44E−08 7.163E−07 LBT-V106 H s DA v FTD n Y t RLRk QLA v KKY l NAI l N Seq ID 27  2.98E−04 5.034E−07 LBT-V107 HS d AVF tDNY t RLR k QLA v KKY l NAI l N Seq ID 28    >1E−6    >1E−6 LBT-V108 HSd AVFTDNY t RLR k QLA v KKY l NAI l N Seq ID 29 LBT-V109 HSD a VFTDNY tRLR k QLA v KKY l NAI l N Seq ID 30 LBT-V110 HSDAV f TDNY t RLR k QLA vKKY l NAI l N Seq ID 31 LBT-V111 HSDAVF t DNY t RLR k QLA v KKY l NAI lN Seq ID 32 LBT-V112 HSDAVFT d NY t RLR k QLA v KKY l NAI l N Seq ID 33LBT-V113 HSDAVFTD n Y t RLR k QLA v KKY l NAI l N Seq ID 34 LBT-V114HSDAVFTDN yt RLR k QLA v KKY l NAI l N Seq ID 35 LBT-V115 HSDAV f TDN ytRLR k QLA v KKY l NAI l N Seq ID 36 LBT-V116 HSDAVF t DNY t RLR k QLA vKKY l NAI l N Seq ID 37 LBT-V117 HSD a VF t DNY t RLR k QLA v KKY l NAIl N Seq ID 38 LBT-V118 HSDAVFTNSY r KVLkRLS a RKL l QDI l Seq ID 39LBT-V119 HSDAVFTNSYR k VLK r LSA r KLL g DIL Seq ID 40 LBT-V120HSDAVFTNS y RKV l KRL s ARK l LQD i L Seq ID 41 LBT-V121 H x DA x FTNSYr KVL k RLS a RKL l QDI l Seq ID 42 LBT-V122 H x DA x FTN xy RKVLK r LSAz KL x QDI l Seq ID 43 LBT-V123 HSDAVFTNSYRKVLK r LSA r KL l QDI l SeqID 44 LBT-V124 HSDAVFTNSYRKVLK r LS a RKL l QD i L Seq ID 45 LBT-V125HSDAVFTNSYRKVLKR l SA r KLL g DI l Seq ID 46 LBT-V126 HfDAVFTDNY t RLR kQLA v KKY l NAI l N Seq ID 47  6.19E−04 3.444E−07 LBT-V127 HfDA x FTDNYt RLR k QLA v KKY l NAI l N Seq ID 48 8.934E−08    >1E−6 LBT-V128 HfDA vFTDNY t RLR k QLA v KKY

NAI l N Seq ID 49 7.118E−08 5.025E−08 LBT-V129 HfDA v FTD n Y t RLR kQLA v KKY l NAI l N Seq ID 50    >1E−6 9.894E−07 LBT-V130 Hf d AVF t DNYt RLR k QLA v KKY l NAI l N Seq ID 51 9.121E−07 6.089E−07 LBT-V131HfDAVFTNSY r KVL k RLS a RKL l QDI l Seq ID 52 LBT-V132 HfDAVFTNSYR kVLK r LSA r KLL g DIL Seq ID 53 LBT-V133 HfDAVFTNS y RKV l KRL s ARK lLQD i L Seq ID 54 LBT-V134 HfDA x FTNSY r KVL k RLS a RKL l QDI l Seq ID55 LBT-V135 HfDA x FTN xy RKVLK r LSA z KL x QDI l Seq ID 56 LBT-V136HfDAVFTNSYRKVLK r LSA r KL l QDI l Seq ID 57 LBT-V137 HfDAVFTNSYRKVLK rLS a RKL l QD i L Seq ID 58 LBT-V138 HfDAVFTNSYRKVLKR l SA r KLL g DI lSeq ID 59 wherein Ac = acetyl; B = AIB; f = D-Phe

In each of sequences above, at least one residue has been optionallyreplaced by a cyclic or heterocyclic β-amino acid residue. In someembodiments, based upon the above sequences, X=ACPC, Z=APC; unchargedside chains replaced by ACPC, basic side chains replaced by APC,Protected β³-amino acids).α/β-Peptide synthesis (Fmoc on the backbonenitrogen and appropriate protecting groups on side chains, whennecessary) will be obtained from commercial suppliers or prepared viareported methods. Each β³-peptide will be prepared manually bymicrowave-assisted Fmoc solid phase peptide synthesis resulting in ac-terminal amide, for example Rink Amide resin. Coupling steps will becarried out with a three-fold excess of the appropriate protected α- orβ³-amino acid, using HATU to mediate amide bond formation. Piperidinewill be used for Fmoc deprotection steps. Each peptide will be cleavedfrom resin by treatment with 94:2.5:2.5 TFA/H2O/triisopropylsilane,precipitated by addition of cold ethyl ether, and purified by reversephase HPLC on a prep-C18 column using gradients between 0.1% TFA inwater and 0.1% TFA in acetonitrile. The identity and purity of the finalproducts will be determined by mass spectrometry and analytical HPLC,respectively.

One purpose of this study is to demonstrate that the analogs of theapplication may be designed to increase the half-life of the polypeptideas compared to the half-life of the naturally encoded protein byintroducing non-natural amino acid analogs that are resistant todegradation and/or induce an equivalent or increased bioactivity ascompared to the naturally encoded polypeptide sequence upon which theanalog is based or derived through the possible incorporation ofconformationally-constrained residues.

Example 6 In-Vitro Cell Based Activity Assay

In Vitro Binding Assay 1: VIP analogs in table 1 were prepared inappropriate phosphate buffer was at pH of 7.5 was exposed to afunctional assay in parallel with wild-type VIP proteins. cAMP Huntercell lines expressing VIPR1 and VIPR2 were expanded from freezer stocksin T25 flasks according to standard procedures and maintained inselective growth media prior to assay. Once it was established that thecells were healthy and growing normally, cells were passaged from flasksusing cell dissociation reagent buffer and seeded into white walledclear bottom 384-well microplates for compound profiling. For profiling,cells were seeded at a density of 10,000 cells per well in a totalvolume of 20 μL and were allowed to adhere and recover overnight priorto compound addition. cAMP modulation was determined using the DiscoveRxHitHunter cAMP XS+ assay.

For profiling compound in agonist mode, the cells were incubated in thepresence of compound at 37° C. for 30 minutes. Cells expressing bothVIPR1 and VIPR2 were exposed to serial dilutions of wild-type VIP andseparate samples of the same type of cells were exposed to serialdilutions of a VIP analogue to determine EC₅₀ values of the analogue ascompared to wild-type VIP. After appropriate compound incubation, assaysignal was generated through incubation with DiscoverX lysis cocktailaccording to the manufacturers standard protocol. Dose curves wereplotted using GraphPad Prism or Activity Base. Percentage activity iscalculated using the following formula:

% Activity=100%×(mean RLU of test sample−mean RLU of vehiclecontrol)/(mean RLU of MAX control−mean RLU of vehicle control).

In Vitro Competitive Binding Assay (prophetic): Binding assays:Membranes prepared from a stable VPAC2 cell line (such as a CHO—S cellline stably expressing human VPAC2 receptor or from cells transientlytransfected with human VPAC1 or PAC1) will be used. A filter bindingassay will be performed using ¹²⁵I-labeled VIP for VPAC1 and VPAC2 and¹²⁵I-labeled PACAP-27 for PAC as the tracers. For this assay, thesolutions and equipment include:

Presoak solution: 0.5% Polyethyleneamine in Aqua destBuffer for flushing filter plates: 25 mM HEPES pH 7.4Blocking buffer: 25 mM HEPES pH 7.4; 0.2% protease free BSAAssay buffer: 25 mM HEPES pH 7.4; 0.5% protease free BSADilution and assay plate: PS-Microplate, U formFiltration Plate Multiscreen FB Opaque Plate; 1.0 mM Type B GlasfiberfilterIn order to prepare the filter plates, the presoak solution will beaspirated by vacuum filtration. The plates will be flushed twice with200 μL flush buffer. 200 μL blocking buffer will be added to the filterplate. The filter plate will then be incubated with 200 μL presoaksolution for 1 hour at room temperature. The assay plate will be filledwith 25 μL assay buffer, 25 μL membranes (2.5 μg) suspended in assaybuffer, 25 μL agonist in assay buffer, and 25 μL tracer (about 40000cpm) in assay buffer. The filled plate will be incubated for 1 hour withshaking. The transfer from assay plate to filter plate will beconducted. The blocking buffer will be aspirated by vacuum filtrationand washed two times with flush buffer. 90 μL will be transferred fromthe assay plate to the filter plate. The 90 μL transferred from assayplate will be aspirated and washed three times with 200 μL flush buffer.The plastic support is removed. It is dried for 1 hour at 60° C. 30 μLMicroscint will beaded. The count will be performed based upon analogaffinity to VPAC1, VPAC2, or PAC1 receptors. IC₅₀ and EC₅₀ calculationswill be performed based upon affinity scoring.

In-vitro Internalization Assay (not prophetic): The analogs of thepresent invention were serially diluted into aqueous solutions withappropriate buffer. The various concentrations of analogs wereadministered to a plurality of cells in culture that expresses relevantnaturally occurring receptor family for the naturally occurringpolypeptide upon which the analog is derived. The analogs wereadministered to the PathHunter® eXpress CHO-K1 VIPR1 (DiscoveRx) cellsaccording to the manufacturers suggested protocol in a 96-well plate.PathHunter® Detection Reagents were used to detect the concentration ofanalog internalized via the endosomal pathway as a function of signalstrength in the absence and presence of wild-type VIP provided as acontrol. Upon receptor internalization, a complete β-galactosidaseenzyme was formed and then able to hydrolyze a DiscoveRx substrate, thusgenerating a chemiluminescent signal. Various EC₅₀ values for the VIPanalogs were calculated per the manufacturer's recommended instructionsusing Graphpad Prism. Chemiluminescent signal were read on a DynexTechnology MLX or BIO-TEK ELx800 Universal Microplate Reader.

FIG. 2: Representative dose response curve for ligand mediated receptorinternalization, resulting in a relative chemiluminescent signal forSeqID 22. EC50 value is 1.024e-008 Molar.

Example 6b Glucose Tolerance Test (not Prophetic)

The analogs described in this document were evaluated in aintraperitinal glucose tolerance test (IPGTT) to determine their abilityto control glucose levels in vivo. 12 DIO mice were fasted overnight andsubsequently received subcutaneous delivery (time point −60) of PBS(n=4), VPAC1 (Seq ID 101, 20 nm/kg, n=3) or VPAC2 (Seq ID 208, 10 nm/kg,n=4) agonist as described herein. Glucose levels were determined using aAviv Accucheck one-touch glucose meter. One hour later, the micereceived a bolus injection of glucose (time-point 0) 1 g/kg and werefollowed for the ensuing 2 hours. Glucose levels were monitored with thesame glucose meter at the following time intervals: 15, 45, 60, 90 and120 minutes after glucose delivery. The data was averaged and plottedusing Graphpad Prism. The VPAC2 agonist evaluated demonstrated modestglucose control when compared to the vehicle arm.

FIG. 9. IP Glucose Tolerance Test for VPAC1 (Seq ID: 101) and VPAC2 (seqid: 208) agonists. DIO mice were fasted overnight followed bysubcutaneous delivery of the specific VIP agonist. 60 minutes later, abolus injection of glucose was delivered intraperitoneally. Glucosereading were taken at the following timepoints −60, 0, 15, 45, 60, 90and 120 minutes post glucose delivery.

Example 7 Structural Analysis of Helical Polypeptides (Prophetic)

This prophetic example describes how the polypeptide analogs of thisinvention may be characterized after manufacture through structuralconformational assays such as circular dichrosim (CD) and Nuclearmagnetic resonance (NMR).

Circular Dichroism Spectroscopy. Circular dichroism measurements will becarried out on an Aviv 202SF Circular Dichroism Spectrophotometer.Samples of each peptide will be prepared with a determined UV absorbancein the range of 0.1-1.0 at 280 nm in a pH buffered solution. Spectrawill be recorded in a 1 mm cell with a step size of 1 nm and anaveraging time of 5 sec. All spectra will be background correctedagainst buffer measured in the same cell. Thermal melts will be carriedout in 1-degree increments with an equilibration time of 2 min betweeneach temperature change. Thermal unfolding data will be fit to a simpletwo state folding model Shortle, D. Meeker, A. K. Freire, E.Biochemistry 1988, 27, 4761-4768) using GraphPad Prism.

Nuclear Magnetic Resonance: Structure elucidation of the proposedanalogs can also be accomplished based on analyses of heteronuclear NMRexperimental data. Global backbone structural information complementingthe local structure information provided by backbone chemical-shiftassignments can be obtained from nuclear Overhauser effect spectroscopy(NOESY) which yield atomic distance constraints together with residualdipolar coupling (RDC) experiments which provide orientation restraintinformation. Together, these techniques can be used to provide valuablestructural information regarding the positioning and alignment of theamino acids within the polypeptide analog. Samples of each peptide oranalog will be prepared with a determined UV absorbance in the range of0.1-1.0 at 280 nm in an appropriate pH buffered solution. Eachpreparation will then be used to determine chemical shifts using thesuite of multidimensional experiments, ie amide based backboneassignments HNCO and TOCSY, followed by conducting structure restraintexperiments, ie NOESY and RDC, using standard NMR equipment (i.e. BrukerNMR) and data analysis software (i.e. Talos+, SPARKY and Al NMR).Further structural insight can be ascertained by comparing the resultsof NMR experiments in the presence and absence of the intended bindingpartner.

One purpose of this study is to evidence that the conformation of theanalog is structurally constrained and that certain non-natural aminoacids have been incorporated in the synthesized peptide in theirpredicted location along a longitudinal axis of the polypeptide.

Example 8 In-Vitro Stability Analysis of Helical Polypeptides inSolution (not Prophetic)

This example describes how the metabolic stability of the polypeptideanalogs of this invention were characterized after manufacture throughassays such as a protease resistance assay.

In Vitro Stability Assay: Stock solutions of the both the naturallyoccurring peptides as well as peptide analogs are prepared at aconcentration of 25 μM (based on UV absorbance) in appropriate buffer. Asolution of proteinase K in addition to other common animal proteases(i.e. Cathepsins, Trypsins, dipeptidyl peptidase IV and chymotrypsin)will be prepared at an appropriate concentration of 50 μg/mL (based onweight to volume) in separate appropriate buffers. For each proteolysisevaluation, 40 μL of peptide stock will be mixed with 10 μL of theappropriate protease stock. The reaction is then allowed to proceed atroom temperature and quenched at the desired time point by addition of100 μL of 1% TFA in water. 125 μL of the resulting quenched reaction arethen injected onto an analytical reverse phase HPLC, and the amount ofstarting peptide present quantified by integration of the appropriatechromatogram peak via absorbance at either 220 or 280 nm. Duplicatereactions are run for each time point. Half-lives are determined byfitting time dependent peptide concentration to an exponential decayusing GraphPad Prism. Samples for some time points will be analyzed bymass spectrometery, and the products observed are used to identify amidebonds cleaved in the course of the reaction. The relative stabilityenhancement is determined through the comparison of the various analogswith its naturally occurring peptide counterpart. Percent degradation isbe quantified by integration of peak areas related to undigested peptidepeaks and corrected for degradation in the absence of enzyme.

FIG. 3: Representative in-vitro protease stability of VPAC-1 selectiveanalog (LBT-V101) compared to the native VIP.

Ex-Vivo Stability Assay (Prophetic):

To investigate the plasma stability of the analogs, both the naturallyoccurring peptide as well as the analogs will be prepared at aconcentration of 100 μM (based on UV absorbance) in appropriate buffer.50 uL aliquots of animal plasma (i.e. rodent, canine, primate) are thenspiked with the analog or the naturally occurring peptide. The reactionwill be allowed to proceed at room temperature and quenched at thedesired time point by addition of an equivalent volume of 1% TFA inAcetonitrile and diluted 1-10 fold with PBS. This solution is thenpassed over a C18 solid phase extraction column (eg. Sigma TPSC18) tofurther isolate the peptide or analog for subsequent LC/MS analysis byremoval of unrelated lipids and plasma proteins. The analogs are theneluted from the C18 column by adding 1-3 column volumes of between 20and 50% acetonitrile, collected and concentrated for subsequentanalysis. Approximately 10 μL of the concentrated quenched reaction willbe injected onto an analytical reverse phase HPLC, and the amount ofstarting peptide present quantified by integration of the appropriatechromatogram peak via absorbance at either 220 or 280 nm. Duplicatereactions will be run for each time point. Half-lives will be determinedby fitting time dependent peptide concentration to an exponential decayusing GraphPad Prism. Samples for some time points will be analyzed bymass spectrometery, and the products observed will be used to identifyamide bonds cleaved in the course of the reaction. The relativestability enhancement will be determined through the comparison of thevarious analogs with its naturally occurring peptide counterpart.

Microsome Stability Analysis of Analogs Polypeptides in Solution(prophetic): Human liver microsomes were prepared by Absorption Systems.A reaction mixture, minus NADPH, was prepared as described below. About1 milligram of the test compound originally in powdered form wassuspended in DMSO prior to addition to a reaction mixture. The testcompound was added into the reaction mixture (0.5 mg/mL human livermicrosomes; 100 mM potassium phosphate; 5 mM Magnesium chloride) at afinal concentration of 1 μM. An aliquot of the reaction mixture (withoutcofactor) was incubated in a shaking water bath at 37° C. for 3 minutes.The control compound, testosterone, was run simultaneously with the testcompound in a separate reaction. The reaction was initiated by theaddition of NADPH cofactor (1 mM NADPH), and the mixture was thenincubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn at 0, 10, 20, 30, and 60 minutes for the test compound and 0,10, 30, and 60 minutes for testosterone. Test compound and testosteronesamples were immediately combined with 400 μL of ice-cold 50/50acetonitrile/dH₂O containing 0.1% formic acid and internal standard toterminate the reaction. The samples were then mixed and centrifuged toprecipitate microsomal proteins. Testosterone samples were assayed byLC-MS/MS using electrospray ionization on a PE SCIEX API 3000 accordingto the manufacturer's instructions. A thermo BDS Hypersil C18 column(30×2.0 mm; 3 μm) was used for chromatography at 300 μL/minute with anaqueous reservoir of 90% water and 10% buffer and an organic reservoirof 90% acetonitrile with 10% buffer (each 25 mM ammonium formate bufferat pH of 3.5). Test compound samples were analyzed by orbitrap. The peakarea response ratio to internal standard (PARR) of the compounds at 10,20, 30, and 60 minutes was compared to the PARR at time 0 to determinethe percent of test compound remaining at each timepoint. After thefinal time point, fluorimetry is used to confirm the addition of NADPHto the reaction mixture. Half-life was normalized of control usinginternal acceptance criteria. Half-life was calculated based upon at½=0.693/k, where k is the elimination rate constant based upon theslope of the plot of natural logarithm percent remaining versusincubation time. Intrinsic clearance (CL_(int)) was calculated basedupon CL_(int)=k/P, where k is the elimination rate constant and P is theprotein concentration in the incubation.

In Vivo Stability Assay (prophetic): To investigate the in vivostability of the analogs, both the naturally occurring peptide as wellas the analogs will be administered to mice and/or rats by IV, IP, SC,PO and/or inhalation routes at concentrations ranging from 0.001 to 50mg/kg and blood specimens withdrawn at 0 minutes, 5 minutes, 15 minutes,30 minutes, 1 hr, 4 hrs, 8 hrs, 12 hrs, 24 hrs and 48 hrspost-injection. Levels of intact compound in 10 uL of fresh plasma willbe injected onto an analytical reverse phase HPLC after following thesolid phase extraction procedure discussed previously, and the amount ofstarting peptide present quantified by integration of the appropriatechromatogram peak via absorbance at either 220 or 280 nm or other meansof measuring the presence or absence of fully intact analog as describedherein. The expected molecular weights will be determined using LC/MSanalysis. This analysis technique also allows the examination of thein-vivo metabolites by determination of fragment molecular weights. Therelative stability enhancement will be determined through the comparisonof the various analogs with its naturally occurring peptide counterpart.

Determination of Oral Bioavailability (prophetic): The oralbioavailability will be evaluated by comparative analysis betweenIntraduodenal and Intrajugular administration of the analogs. Eachcompound/test article will be converted to an appropriate salt form anddissolved in a combination of biocompatible acids (e.g. citric acid,citrate, taurodeoxycholic acid, stearic acid, etc.) along with anoptional permeation enhancer (e.g. DL-Lauroylcarnitine, sodium laurylsulfate, Acetylated monoglycerides, sucrose, etc).

Intraduodenal and Intrajugular catheterized male C57/BL6 mice, 8 weeksold (Charles River Labs) are quarantined for at least one day and havecontinuous access to food and water. A single dose of the appropriateanalog is then administered via catheter to each mouse. The dose (1-1000nm/kg) and time will then be recorded.

Blood samples will be collected via tail vein at the following timepoints: 0 h, 0.5 h, 1 h, 3 h, 6 h and 12 h. Samples will be kept in EDTAcontaining (10 uL of 50 mM) microtainer tubes under subambienttemperature (4° C.) before they are processed. Blood samples will becentrifuged (10,000 rpm for 5 minutes) and plasma samples should beremoved and stored in a −20° C. freezer until analyzed for analoglevels. Analog levels in the plasma will be analyzed using the followingprotocol for direct plasma precipitation.

The in vivo plasma samples will be prepared in a 1.5 mL 96-well plate,by adding, in order, 100 μL of the test plasma, 150 μl of methanol,followed by vortexing for 10-20 seconds. 150 μL of 0.05 ng/μL of anInternal Standard in acetonitrile shall be added and vortexed for 30seconds.

The standard curve samples were prepared in a 1.5 mL 96-well plate, byadding, in order, 1004 of control mouse plasma, followed by 1504 ofmethanol and vortexing for 10-20 seconds. 1504 of 0.05 ng/μL of anInternal Standard in acetonitrile shall be added and vortexed for 30seconds. The samples will then be spiked with 0-200 ng (10concentrations) of the compound of interest in 50% methanol to obtain astandard curve range of 0.5 ng/mL to 2,000 ng/mL. Again, the sample isvortexed for 30 seconds.

The samples should then be centrifuged for 20-30 minutes at 3,000 rpm inan Eppendorf microfuge before 80-90% of supernatant is transferred intoa clean 96-well plate. The organic solvent will then be evaporated untilthe samples are dry (under N₂ at 40° C./30-60 min. (ZymarkTurbovap)).

The residue will then be dissolved in <100 uL mobile phase (40%CH₃OH/0.1% TFA). LC/MS/MS will then be run using a mass spectrometerwith pump. Data analysis and quantification accomplished using ThermoLCQ ion trap mass spectrometer. A 5-50 μl sample volume will be injectedonto a reverse phase column (Keystone 2.0×20 mm, 5 μm, PN: 8823025-701)using a mobile phase of 40% CH₃OH, 0.1% TFA-100% CH₃OH, 0.1% TFA. Therun time will be about 8 minutes at a flow rate of about 400 μL/minutes.The Area Under the Curve (AUC) will be calculated using the lineartrapezoidal rule from t=0 to the last plasma concentration sampling timetx (see Handbook of Basic Pharmacokinetics, Wolfgang A. Ritschel andGregory L. Kearns, 5th ed, 1999).AUC⁰-tx=.SIGMA.⁰-n((C_(n)+C_(n)+1)/2))(t_(n)+1−t_(n)) {in (μg/mL)h}

One purpose of this study is to evidence that the analog is moreresistant to peptidases as compared to the resistance ofsimilarly-structured, naturally occurring polypeptides upon which thestructure of the analog is based or derived. The results may show that,when treated with the same proteolytic enzymes, the analogs of theinvention will resist degradation and have longer half-lives thansimilarly-structured, naturally occurring polypeptides upon which thestructure of the analog is based or derived.

Example 9 In-Vivo Functional Analysis of VPAC1 Selective Polypeptides(Prophetic)

This prophetic example describes the function of polypeptide analogs ofthis invention may be characterized after manufacture through assaysthat measure bioactivity of the analogs when exposed to tissue cultureor when administered to an animal model of one of the following humandisease states: Acute respiratory distress, chronic obstructed pulmonarydisorder, asthma, chronic obstructive pulmonary disease, sarcoidosis,small lung cell cancer, exocrine pancreatic tumors, colorectalcarcinoma, gastric carcinoma, hepatocellular carcinoma, esophagealcarcinoma, renal cell carcinoma, prostate carcinoma, urinary bladdercarcinoma, liver carcinoma, ductal pancreatic cancer, breast carcinoma,ovarian carcinoma, non-hodgkin's lymphoma, meningioma, GEP tumors(differentiated and undifferentiated), pituitary adenoma, endometrialcancer, astrocytoma, giloblastoma, non-small cell lung cancer,pancreatic cancer, melanoma, renal cancer, neuroblastoma, leukima andprostate cancer, autoimmune disease, inflammatory disease, sepsis,Hirschsprung's Disease, sexual dysfunction, erectile dysfunction,Parkinson's disease, Alzheimer's disease, circadian rhythm dysfunction,pain, colorectal cancer, hepatocellular cancer, elevated blood glucoselevels, hyperglycemia, diabetes, insulin resistance, metabolic acidosis,obesity, Type I diabetes, Type II diabetes, Multiple Sclerosis,osteoporosis, Sjogren's syndrome, pancreatitis, uveoretinitis,osteoporosis, female sexual dysfunction and diabetic nephropathy.

In Vivo Efficacy in Animal Models: To determine the activity of analogsof the invention in vivo as compared to the naturally occurringpolypeptides upon which the analogs are derived, the analogs will beadministered alone (IP, IV, SC, PO, by inhalation or nasal routes) or incombination with known active agent to monitor the above-mentioneddisease states. Secretin family analogs alone or in combination withsub-optimal doses of relevant active agents for specific indications ordisease states will be, for example, administered to an appropriateanimal model mice (8-10 days after injection/day 1 of experiment) bytail vein or IP routes at doses ranging from 0.0001 mg/kg to 50 mg/kgfor 1 to 21 days. Optionally, the mice will be assayed throughout theexperiment with a selection marker relevant to the particular studiesdisease state every other day and survival monitored daily for theduration of the experiment. Expired mice will be optionally subjected tonecropsy at the end of the experiment. These in vivo tests optionallygenerate preliminary pharmacokinetic, pharmacodynamic and toxicologydata.

Adjuvant-Induced Arthritis in Rats: Adjuvant induced arthritis (“AIA”)is an animal model useful in the study of rheumatoid arthritis (“RA”),which is induced by injecting M. tuberculosis in the base of the tail ofLewis rats. Between 10 and 15 days following injection, animals developa severe, progressive arthritis.

Generally, analogs will be tested for their ability to alter hind pawswelling and bone damage resulting from adjuvant induced edema in rats.To quantitate the inhibition of hind paw swelling resulting from AIA,two phases of inflammation have been defined: (1) the primary andsecondary injected hind paw, and (2) the secondary uninjected hind paw,which generally begins developing about eleven days from the inductionof inflammation in the injected paw. Reduction of the latter type ofinflammation is an indication of immunosuppressive activity. Cf. Chang,Arth. Rheum., 20, 1135-1141 (1977).

Using an animal model of RA, such as AIA, enables one to study thecellular events involved in the early stages of the disease. CD44expression on macrophages and lymphocytes is up regulated during theearly development of adjuvant arthritis, whereas LFA 1 expression is upregulated later in the development of the disease. Understanding theinteractions between adhesion molecules and endothelium at the earlieststages of adjuvant arthritis could lead to significant advances in themethods used in the treatment of RA.

Collagen Induced Arthritis in Rats: To determine the efficacy of arepresentative analog of this invention administered by po bid dosing(Days (−1)-20) for inhibition of the inflammation, cartilage destructionand bone resorption that occurs in developing type II collagen arthritisin rats.

Animals: Female Lewis rats (Harlan), weighing 125-150 g on arrival.(inject subtotal of rats with collagen to get responders on days 10, 11,12 for 6 groups of 10). The animals (a group for arthritis, a group fornormal control), housed 4-5/cage, will be acclimated for 4-8 days. Theanimals will be dosed from about po1 mg/kg bid to po100 mg/kg bid.

Materials: Peptides or analogs in vehicle, Type II collagen, Freund'sincomplete adjuvant, methotrexate (Sigma)

General Study Design: Dosing initiated on day minus 1. The acclimatedanimals will be anesthetized with isoflurane and given collageninjections (D0). On day 6 they will be anesthetized again for the secondcollagen injection. Collagen is prepared by making a 4 mg/mL solution in0.01 N acetic acid. Equal volumes of collagen and Freund's incompleteadjuvant, will be emulsified by hand mixing until a bead of thismaterial held its form when placed in water. Each animal will receive300 uL of the mixture each time spread over 3 sites on back. Caliperingof normal (pre-disease) right and left ankle joints are to be doneapproximately one ay prior to the expected days on onset of disease.

Rats will be weighed on days (−) 1, 6, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, and 20 of the study and caliper measurements of ankles takenevery day beginning on day 9. Final body weights will be taken on day20. After final body weight measurement, animals are to be anesthetizedfor terminal plasma collection and then euthanization. Both hind pawsand knees will be removed. Hind paws will be weighed, placed (withknees) in formalin and then processed for microscopy.

Processing of Joints: Following 1-2 days in fixative and then 4-5 daysin decalcifier, the ankle joints will be cut in half longitudinally,knees will be cut in half in the frontal plane, processed, embedded,sectioned and stained with toluidine blue.

Induction of Colitis in HLA-B27 Rats: The efficacy of the analogs of thepresent invention in reversing colitis can be determined in HLA-B27transgenic rats. HLA-B27 transgenic rats have been utilized as an animalmodel of Inflammatory Bowel Disease which mimics Crohn's Disease inhumans. The rats overexpress the human MHC class I HLA-B27 heavy chainand beta-2 microglobulin proteins, which induces a variety of autoimmunediseases that include inflammation of the colon.

The therapeutic effect of the analogs described in this invention interms of resolving colitis can be evaluated in HLA-B27 transgenic rats.Diseased rats will be dosed subcutaneously with 0.001-100 mg/kg of asingle analog of this invention once or twice a day for 16 days or onceper week for two weeks.

Disease Activity Index (DAI) scores will be used to determine theefficacy of each analog as compared to rats dosed with vehicle. Inaddition, fecal consistency and FOB scores for both rats dosed withanalogs will be statistically compared to the vehicle group.

Induction of Colitis: 1-20 HLA-B27 (6-9 weeks old) transgenic rats willbe acclimated in animal facility for 10 weeks Animal bedding will bemixed from different cages once a week to control for a “dirty”environmental flora.

Treatments: Rats are to be enrolled and randomized into four groups(n=5) based on weight and DAI scores (FC.gtoreq.3, FOB.gtoreq.2). Theexperimental groups will be dosed subcutaneously with an analog0.001-100 mg/kg once or twice a day for 16 days or once per week for twoweeks and terminated at trough. The control groups include avehicle-treated group and a GG5/3 (mouse anti-rat alpha-4 integrinantibody) positive control group dosed subcutaneously at 10 mg/kg (5mL/kg) on d0, d3, and d6 and terminated at trough on d8. Fresh analogand vehicle treatments are to be formulated in advance of treatment.

Endpoint Read-outs: Disease Activity Index scores, Fecal Consistencytest and Fecal Occult Blood test, are to be taken 4 times a week togenerate in-life clinical scores. The primary read-out for the study isa histopathological analysis of cecum, proximal colon, mid-colon, anddistal colon. An IBD scoring system was applied (Table H2). TABLE H2 IBDScoring System Multiple Endpoints A Destruction of epithelium and glandsB Dilatation of glandular crypts C Depletion and loss of goblet cells DInflammatory cell infiltrates E Edema F Vascular congestion G CryptAbscesses H Atrophia

Parkinson's Disease Model: Male C57/BL6 mice, 8-12w old, housed in theIACUC approved housing are administered an analog described herein aswell as a pharmaceutically acceptable carrier for five days at a dosebetween (0.1 nM and 100 nM). After five days, SPC and Tregs from themice are harvested and then transferred to another set of MPTPintoxicated mice using the following protocol.

Five days following boost, mice were sacrificed, and single-cellsuspensions were prepared from inguinal lymph nodes and spleens. CD4+ Tcell populations from spleens and lymph nodes were enriched by negativeselection with CD4-enrichment columns (R&D Systems, Minneapolis, Minn.),followed by CD25-PE positive selection with AutoMACS (Miltenyi Biotec,Auburn, Calif.). As determined by flow cytometric analysis, populationsof Tregs and Teffs were consistently 0.95% pure using this method (12).T cells were cultured in complete RPMI 1640 (RPMI 1640 [Invitrogen,Carlsbad, Calif.] supplemented with 10% FBS, 2 mM L-gluta-mine, 25 mMHEPES, 1 mM sodium pyruvate, 13 nonessential amino acids, 55 nM 2-ME,100 U/ml penicillin, and 100 mg/ml streptomycin [Mediatech, Manassas,Va.]) in the presence of anti-CD3 (145-2C11; BD Pharmingen, San Diego,Calif.), 4YSyn, or N-4YSyn. Proliferation and inhibition assays wereperformed, as described (3, 10). MPTP-intoxicated mice received an i.v.tail injection of 5 3 107 freshly isolated SPCs or 1 3 106 freshlyenriched Tregs in 0.25 ml HBSS. Each Th subset was harvested, and 10 3106 T cells from each subset were transferred to separate recipientgroups. For stimulation of cytokine production, Th subsets werestimulated with 20 ng/ml PMA and 1 mM ionomycin (Sigma-Aldrich) for 5 h,cells were washed, media were replaced, and supernatants were collected24 h later for analysis. (J Immunol 2010; 184:2261-2271).

Recipient mice are then followed and sacrificed, their brains areexcised and stained with appropriate markers for microglia cellidentification according to the following protocol:

Mice were postmortem transcardially perfused with PBS, followed by 4%para-formaldehyde (Sigma-Aldrich). Frozen midbrain sections (30 mm) wereimmunostained for Mac-1 (CD11b, 1:1000; Serotec, Raleigh, N.C.).Fluorojade C (FJ-C) staining (Millipore, Billerica, Mass.) was performedon adjacent sections, according to the manufacturer's protocol, toassess de-generating neurons and was quantified using ImageJ. Overall,dopaminergic neuron survival was assessed 7 d following MPTPintoxication and resolution of cell death processes with polyclonal Absto mouse tyrosine hydroxylase (TH; 1:1000; EMD Chemicals/Calbiochem, SanDiego, Calif.) and were counterstained for Niss1 substance by thioninstaining Total numbers of Mac-1+ cells, CD4+ T cells, and TH- andNiss1-stained neurons in the SN were estimated by stereo-logicalanalysis with Stereo Investigator software (MBF Bioscience, Williston,Vt.), using the optical fractionator module. Quantitation of striatal TH(1:500; EMD Chemicals/Calbiochem, Gibbstown, N.J.) was performed bydensitometric analysis. Adjacent midbrain sections were immunostainedfor CD4 (clone RM4-5, 1:200, BD Pharmingen). Sections were incubated instreptavidin-HRP solution (ABC Elite vector kit, Vector Laboratories,Burlingame, Calif.) and color developed using a generation systemconsisting of diaminobenzidine (DAB) chromogen (Sigma-Aldrich). (JImmunol 2010; 184:2261-2271).

Comparisons between treated and untreated brain sections are conductedand evaluated for neuroprotection and neuroregeneration. Brain sectionimages can be analyzed for stain density and location to arrive at astatistical performance evaluation

In-vivo Cancer Model: Female athymic BALByc nude mice, 4-5 weeks old,will be housed in filter-top cages in a pathogen free,temperature-controlled, laminar-flow, filtered-air, isolated room andwill be exposed to light from 7:00 a.m. to 7:00 p.m. Cells from theappropriate tumor type will be injected subcutaneously into the rightflank of each mouse. There were four experimental groups, of four miceeach, three of which will receive VIP and/or an analog of VIP (1.0, 5.0,or 10 mg/day) in PBS; as a control, the fourth will receive only PBS.All solutions will be infused for 8 weeks, beginning 1 week afterinjection of the cells, and delivered by i.v., i.p., subc., i.m.injection or osmotic pumps placed aseptically under the skin of the backof the mice. The pump will release its contents at a rate of 0.5 ml/hfor a duration of 2 weeks. The spent pumps will be removed every 2weeks, and new pumps, containing fresh solutions, will be implanted withknown techniques; this procedure will be repeated three times. Aftertreatment, the tumors will be measured with calipers, and the mice willbe weighed weekly for 8 weeks. Tumor volume will be calculated for anellipsoid as (maximal length)×(maximal height)×(maximal width)×(R/6). Onthe last day of the experiment, blood will be sampled from theretroorbital plexus into chilled heparin-containing tubes rinsed with0.05% NaEDTA and containing three protease inhibitors, 10 mg/ml soybeantrypsin inhibitor, 100 TIU/ml aprotinin, and 10 mg/ml phosphamidon), aswell as 0.1 mM IBMX for measurement of plasma VIP and cAMP levels. Themice will then euthanized. The tumors will be excised, weighed, andfrozen in liquid nitrogen for subsequent extraction (in methanol) andfor measurement of protein content by known techniques; a portion of thetumor will be fixed in 10% neutral buffered formalin for morphologicexamination.

One purpose of these studies is to evidence that the analogs are capableof producing the desired biological, biochemical, diagnostic, medicinaland/or therapeutic outcome in a living animal.

Example 10 (Prophetic) In Vivo Functional Determination in DiabeticNephropathy Models

In order to determine if the analogs presented herein are capable ofbeneficial effects in animal models designed to mimic human diabeticnephropathy. An example of one of the appropriate protocols is describedbelow and would be familiar to those skilled in the art. BTBR ob/ob micetreatment and analysis. BTBR ob/ob mice along with heterozygous BTBRob/+ control mice will be purchased from Jackson Laboratories. Mice willbe injected subcutaneously with 1 pM/kg to 1 mM/kg of VIP analog orsaline as reported previously, daily, three times per week, once perweek or less frequently for up to 5 months. Urine will be collected, andbody weight and glycemia (OneTouch) will be determined weekly.Approximately six mice per group will be analyzed. All animal procedureswill be approved by the Institutional Animal Care and Use Committee(TACIT). After isotonic saline perfusion, the right kidney will beremoved for cholesterol content determination and mRNA extraction. Oneleft kidney pole will be embedded in OCT, while a second pole will befixed in 4% PFA and paraffin-embedded for histological analysis. Bloodsamples will be analyzed for CBC, lipid panel, AST, ALT, AlkalinePhosphatase, GGT, and BUN in a Laboratory Core Fac 1 such as the onelocated at University of Miami. Serum creatinine can be determined bytandem mass spectrometry at an appropriate facility, using the methodspreviously described. The urine albumin content can be measured by ELISA(Bethyl Laboratories). Urinary creatinine will be assessed by an assaybased on the Jaffe method (Stanbio). Values will be expressed as igalbumin/mg creatinine. Fasting plasma insulin can be determined by ELISA(Mercodia, SW). intraperitoneal glucose tolerance tests (IPGTT) will beperformed up to 4 months after treatment onset; after an approximate5-hr fasting, blood glucose will be recorded at baseline and then up to180 minutes after a glucose bolus (1.5 g/kg). For insulin sensitivity,glycemia will be monitored at baseline and up to 150 minutes afterintraperitoneal injection of 4 mU/g of short acting insulin. All studieswill be conducted in a head-to-head manner to determine functionalperformance and the analogs described herein would be compared tostandard ACE (angiotensin converting enzyme) inhibitors and ARBs(angiotensin receptor blockers) such as (Lotensin, Capoten, Vasotec,Monopril, Atacand, Teveten, Cozaar Diovan, etc).

Histology, assessment of mesangial expansion and glomerular surfacearea. Periodic acid-Shiffs (PAS) staining of approximately 4 μm thicktissue sections will be performed. Approximately twenty glomeruli persection will be analyzed for mesangial expansion by semiquantitativeanalysis (scale 0-4) and would be performed by two blinded independentinvestigators. The glomerular surface will be delineated in eachencountered glomerulus and the mean surface area will be calculated.

The following journal articles, which are herein incorporated byreference, disclose VIP family analogs contemplated to be a polypeptidebackbone for the VIP family analogs of the invention. The journalarticles also disclose a series of methods of administering VIP familyanalogs as part of pharmaceutical compositions:

-   1. Gozes, et. al., Current Pharmaceutical Design, 2003, Vol. 9, No.    6-   2. Delgado, et. al., Brain Behav Immun. 2008 November; 22(8):    1146-1151. doi:10.10166.bbi.2008.06.001.-   3. L. Dickson, K. Finlayson/Pharmacology & Therapeutics 121 (2009)    294-316.-   4. Gonzales-Rey, et. al., TRENDS in Pharmacological Sciences Vol. 28    No. 9.-   5. Varela, et. al., Expert Opin. Biol. Ther. (2007) 7(4):461-478-   6. Brenneman, Peptides 28 (2007) 1720-1726;-   7. Onoue, et. al., Naunyn-Schmiedeberg's Arch Pharmacol (2008)    377:579-590

Any journal article, patent application, issued patent or otherpublication referenced in this application is herein incorporated byreference. The embodiments listed herein are not meant to berestrictive, but rather illustrative of the invention.

1. A composition comprising at least one vasoactive intestinal peptide(VIP) analog comprising HSDAVFTDNYTRLRKQMAVKKYLNSILN wherein at leastthree amino acids of SEQ ID NO:1 are replaced with a cyclic beta-3 aminoacids and wherein the analog comprises an amino acid sequence thatselectively binds to VIP Receptor
 1. 2.-5. (canceled)
 6. The compositionof claim 1, wherein the analog is derived from a human VIP amino acidsequence. 7.-13. (canceled)
 14. The composition of claim 1, wherein theβ-amino acids are spatially aligned along a longitudinal axis of theanalog in order to constrain the conformation of the analog in an activestate.
 15. The composition of claim 1, wherein the ratio of totalβ-amino acids to amino acids in the analog is from 1 to 3 β-amino acidsfor every 7 amino acids of the analog.
 16. (canceled)
 17. Thecomposition of claim 1, wherein the analog does not comprise arepetitive pattern of sequential β-amino acids from the amino-terminusto the carboxy-terminus selected from the following: ααααααβ, αααααβα,ααααβαα, αααβααα, ααβαααα, αβααααα, βαααααα, αααααββ, ααααββα, αααββαα,ααββααα, αββαααα, ββααααα, βαααααβ, βααααβα, βαααβαα, βααβααα, βαβαααα,αβααααβ, αβαααβα, αβααβαα, αβαβααα, ααβαααβ, ααβααβα, ααβαβαα, αααβααβ,αααβαβα, and ααααβαβ,
 18. The composition of claim 1, wherein the analogcomprises a repetitive pattern of β-amino acids from the amino-terminusto the carboxy-terminus.
 19. A pharmaceutical composition, comprising:(i) the analog of claim 1, or a pharmaceutical salt derived therefrom;and (ii) a pharmaceutically acceptable carrier.
 20. The pharmaceuticalcomposition of claim 19, wherein the composition further comprises oneother active agent.
 21. A method of manufacturing the pharmaceuticalcomposition of claim 19 comprising catalyzing a reaction between atleast one α-amino acid with at least one β-amino acid.
 22. A method oftreating or preventing pulmonary hypertension, primary arterialhypertension (PAH), pulmonary hypertension associated topost-ventricular septal defect, idiopathic pulmonary fibrosis,idiopathic pulmonary arterial hypertension, high blood pressure, CRESTsyndrome—Calcinosis; Raynaud's disease; loss of muscle control of theEsophagus; Sclerodactyly; Telangiectasia, Acute respiratory distress,congestive heart failure, chronic obstructed pulmonary disorder, asthma,chronic obstructive pulmonary disease, sarcoidosis, small cell lungcancer, autoimmune disease, inflammatory disease, sepsis, Hirschsprung'sDisease, sexual dysfunction, erectile dysfunction, Parkinson's disease,Alzheimer's disease, circadian rhythm dysfunction, pain, colorectalcancer, hepatocellular cancer, elevated blood pressure levels, elevatedblood glucose levels, elevated blood pressure levels, hyperglycemia,diabetes, insulin resistance, metabolic acidosis, obesity, Type Idiabetes, Type II diabetes Multiple Sclerosis, osteoporosis, Sjogren'ssyndrome, pancreatitis, uveoretinitis, osteoporosis, female sexualdysfunction in a subject in need thereof comprising administrating thepharmaceutical composition of claim
 19. 23. A method of inhibitingsecretion of TNF-α in a subject comprising administering a compositioncomprising a VIP analog to a subject, wherein said analog comprises anα-amino acid and at least one β-amino acid.
 24. A method of identifyinga modulator of human VIP receptor activity comprising: a) contacting ahuman VIP receptor with a VIP analog, wherein said analog comprises anα-amino acid and at least one β-amino acid b) measuring the associationof the VIP analog to the human VIP receptor in the presence and absenceof an unknown compound and c) comparing the rate of association of theVIP analog to the human VIP receptor in the presence of an unknowncompound to the rate of association of the VIP analog to the human VIPreceptor in the absence of an unknown compound.
 25. A kit comprising afirst container comprising the pharmaceutical composition of claim 19.26. The kit of claim 25, further comprising a second containercomprising a vehicle for administration of the composition of claim 1.27.-28. (canceled)
 29. The composition of claim 1, wherein the VIPanalog comprises an amino acid sequence at least 70% homologus to anamino acid sequence disclosed in Table
 1. 30.-31. (canceled)
 32. Thecomposition of claim 29, wherein the total number of β-amino acids inthe analog is from about 30 percent to about 50 percent of the totalnumber of amino acids of the analog. 33.-46. (canceled)
 47. Thecomposition of claim 29, wherein said analog comprises an α-amino acidand at least two β-amino acids and does not comprise any repeatingpattern of alpha and beta amino acids, except a pattern of alpha andbeta amino acids comprising: αααβ, and wherein a represents an alphaamino acid and β represents a beta amino acid.
 48. (canceled)
 49. Amethod of treating cancer comprising administering a prophylactically ortherapeutically effective amount of a pharmaceutical composition ofclaim 19, wherein the cancer is chosen from one or more of the followingtypes: small lung cell cancer, exocrine pancreatic tumors, colorectalcarcinoma, gastric carcinoma, hepatocellular carcinoma, esophagealcarcinoma, renal cell carcinoma, prostate carcinoma, urinary bladdercarcinoma, liver carcinoma, ductal pancreatic cancer, breast carcinoma,ovarian carcinoma, non-hodgkin's lymphoma, meningioma, GEP tumors(differentiated and undifferentiated), pituitary adenoma, endometrialcancer, astrocytoma, giloblastoma, non-small cell lung cancer,pancreatic cancer, melanoma, renal cancer, neuroblastoma, leukima andprostate cancer. 50.-51. (canceled)